Near-infrared fluorescent dyes with large stokes shifts

ABSTRACT

Embodiments of near-infrared (NIR) dyes are disclosed, along with methods and kits for detecting analytes with the NIR dyes. The NIR dyes have a structure according to the general structure 
                         
At least one of R 1 /R 2 , R 2 /R 3 , R 3 /R 4 , R 5 /R 6 , R 6 /R 7 , and/or R 7 /R 8  together forms a substituted or unsubstituted cycloalkyl or aryl.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/129,223, filed Dec. 24, 2013, now issued as U.S. Pat. No. 9,250,246,which is the National Stage of International Application No.PCT/US2012/045116, filed Jun. 29, 2012, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 61/505,038, filed Jul. 6, 2011, and U.S.Provisional Application No. 61/502,839, filed Jun. 29, 2011, each ofwhich is incorporated herein by reference in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under R01 EB002044awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD

Embodiments of near-infrared (NIR) dyes, methods, and kits for detectinganalytes are disclosed.

BACKGROUND

A barrier to simplified diagnostic testing is that current clinicalchemistry technologies require significant sample preparation andhandling for the analysis of complex biological samples. Samplepreparation is a major bottleneck in diagnostics. Indicator fluorophoresfor specific biomarkers capable of functioning directly in an analyte'smedium (e.g., blood, urine) without sample handling or separation stepswould require fewer manipulations, thereby producing quicker results andreducing potential health hazards due to sample handling. However,surprisingly little progress has been made in developing suchfluorophores. This is due at least partially to the relative lack oflong wavelength probes.

There are relatively few classes of near infrared (NIR) active dyes thatare routinely used, and only one NIR dye is currently approved forclinical use. Advantages of NIR dyes include minimal interferingabsorption and fluorescence from biological samples, inexpensive laserdiode excitation, and reduced scattering and enhanced tissue penetrationdepth. However, there are only relatively few classes of such dyesreadily available. These include the phthalocyanines, cyanine dyes andsquaraine dyes. Each class of dye has inherent strengths andlimitations. For example, almost all the established groups oflong-wavelength fluorophores have very small Stokes shifts (i.e.,emission-excitation wavelength differences), e.g., 10 nm (Miller,Springer Ser. Fluoresc., 2008, 5, 147-162). If used in conjunction witha relatively broad band light source, such as an LED, there may besignificant scattered light background signal, producing a poorsignal:noise ratio.

Previous research has investigated red-shifting xanthene dyes forbiodiagnostics and imaging applications. Long-wavelength, xanthene-baseddyes have been used in cellular imaging applications. However, theirspectral properties do not fall within the useful NIR “blood window” of700-800 nm, which facilitates analyte detection in blood. Rhodamines are“red” or long-wavelength xanthene dyes. One notable long wavelengthxanthene dye is rhodamine 800, which emits at the interface of the redand NIR, a few nanometers above or below 700 nm depending on thesolvent. However, it suffers from limited water solubility and dimerformation and a small Stokes shift of 16 nm (Sauer et al., J. Fluoresc.,1995, 5, 247-261), which complicates analysis in blood. Anotherinnovation includes “JA” dyes, which shift the spectra toward longerwavelength through the addition of double bonds to thenitrogen-containing rings. (Sauer et al.; U.S. Pat. No. 5,750,409).Arden-Jacob and co-workers developed an improved series of fluorophoresfor biodiagnostics in the red region. However, these dyes exhibit rathersmall Stokes shifts and do not absorb or emit in the NIR (U.S. Pat. No.5,750,409).

Annulation is another approach used to produce longer wavelengthfluorophores. Type [c] annulated xanthenes includeseminaphthofluorescein (SNAFL) and seminaphthorhodafluor (SNARF)compound developed by Haugland (Whitaker et al., Anal. Biochem., 1991,194, 330-344), which have been used as ratiometric pH sensors, metal ionsensors and imaging probes. (Chang et al., PNAS, 2004, 101, 1129-1134;Nolan et al. J. Am. Chem. Soc., 2007, 129, 5910-5918.)

SUMMARY

Embodiments of compounds having emission spectrum maxima in thenear-infrared region (near-infrared (NIR) dyes) are disclosed. Someembodiments of the disclosed compounds exhibit large bathochromic shiftsand/or enhanced Stokes shifts compared to currently available NIR dyes.Embodiments of the disclosed compounds have a structure according togeneral formula I.

In general formula I, each bond depicted as “

” is a single or double bond as needed to satisfy valence requirements.X¹ is O, S, Se, Si(CH₃)₂, Ge(CH₃)₂, Sn(CH₃)₂, CH₂, C(CH₃)₂ or NH; R¹,R², and R⁴ independently are hydrogen, hydroxyl, oxygen, thiol, loweralkyl, carboxyalkyl, amino, substituted amino, alkoxy, halogen, or—NHR^(c) where R^(c) is

R⁵, R⁷, and R⁸ independently are hydrogen, hydroxyl, thiol, lower alkyl,carboxyalkyl, amino, substituted amino, alkoxy, or halogen; R³ and R⁶independently are hydrogen, hydroxyl, halogen, oxygen, sulfur, thiol,amino, alkyl amino, imino, iminium, alkyl imino, alkyl iminium,cycloalkyl imino, or —NHR^(c) where R^(c) is as defined above; and atleast one of R¹ and R², R² and R³, R³ and R⁴, R⁵ and R⁶, R⁶ and R⁷,and/or R⁷ and R⁸ together form a substituted or unsubstituted cycloalkylor aryl. R⁹-R¹² independently are hydrogen, alkyl, acyl, carboxyl,nitro, amino, alkyl amino, or —SO₃H. R¹³ is hydrogen, hydroxyl, loweralkyl, lower alkoxy, —SO₃H or —COOR¹⁴ where R¹⁴ is hydrogen or loweralkyl and the bond depicted as “

” in ring B is a double bond, or R¹³ is one or more atoms forming a ringsystem with rings B and D and the bond depicted as “

” in ring B is a single bond.In some embodiments, the compounds have a chemical structure accordingto general formulas (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), or(XI)

where each bond depicted as “

” is a single or double bond as needed to satisfy valence requirements;X¹, R¹-R⁵ and R⁷-R¹⁴ independently are as defined above; R⁶ is hydrogen,hydroxyl, halogen, thiol, amino, alkyl amino, or —NHR^(c) where R^(c) isas defined above when the bond between R⁶ and ring A is a single bond,or R⁶ is oxygen, sulfur, imino, iminium, alkyl imino, alkyl iminium, orcycloalkyl imino when the bond between R⁶ and ring A is a double bond;and R¹⁵-R²² independently are hydrogen, halogen, hydroxyl, oxygen,thiol, amino, alkyl amino, alkoxy, sulfur, imino, iminium, alkyl imino,alkyl iminium, or —NHR^(c) where R^(c) is as defined above; and However,if X¹ is oxygen in general formula (III), then R⁶ is other than oxygenor alkyl amino, or R¹³ is other than one or more atoms forming a ringsystem with rings B and D, or R¹⁶ is other than hydroxyl or hydrogen, orR¹⁸ is other than hydroxyl or hydrogen, or at least one of R¹, R², R⁵,R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², or R¹³ is other than hydrogen; if X¹ isoxygen, sulfur, CH₂, or NH in general formula (IV), then at least one ofR¹, R², R⁷-R¹³, or R¹⁵-R²² is other than hydrogen, hydroxyl, halogen,oxygen, lower alkyl, amino, or thiol, or R¹³ is one or more atomsforming a ring system with rings B and D and the bond depicted as “

” in ring B is a single bond; if X¹ is oxygen and R¹⁵ or R¹⁷ is hydroxylin general formula (V), then R⁶ is other than oxygen, amino, or alkylamino, or at least one of R¹, R⁸, or R¹⁸ is other than hydrogen, or R¹³is one or more atoms forming a ring system with rings B and D and thebond depicted as “

” in ring B is a single bond; or if X¹ is oxygen and either R¹⁶ or R¹⁸is hydroxyl in general formula (VII), then R⁶ is other than oxygen,amino, or alkyl amino, or at least one of R³, R⁸ or R¹⁵ is other thanhydrogen, or R¹³ is one or more atoms forming a ring system with rings Band D and the bond depicted as “

” in ring B is a single bond.

In some embodiments, X¹ is oxygen. In certain embodiments, R¹³ is —COO—and forms a lactone ring. In some embodiments, the compound comprises atleast one halogen atom positioned adjacent to an ionizable moiety.

In some embodiments, compounds according to general formulas (III)-(XI)have emission spectrum maxima at a wavelength greater than or equal to700 nm, or greater than or equal to 750 nm. In certain embodiments, thecompounds have a Stokes shift greater than or equal to 80 nm, such asgreater than or equal to 100 nm.

In some embodiments, the compounds have a chemical structure accordingto general formula (III) where R¹, R², R⁵, R⁷, and R⁸, are hydrogen orhalogen; R⁶ is oxygen, imino, iminium, lower alkyl iminium, or —NHR^(c)where R^(c) is as defined above, and R⁹-R¹² independently are hydrogen,amino, lower alkyl, carboxyl, or —SO₃H. In certain embodiments, thecompounds have a chemical structure according to general formula (III)where R¹, R², R⁵, R⁷-R¹², and R¹⁵-R¹⁷ are hydrogen, R⁶ is iminium or—NHR^(c) where R^(c) is as defined above, R¹³ is —COOR¹⁴ where R¹⁴ ishydrogen or lower alkyl, and R¹⁶ and R¹⁸ independently are amino or—NHR^(c) where R^(c) is as defined above.

In some embodiments, the compounds have a chemical structure accordingto general formula (IV), (IX), or (X) where X¹ is oxygen and R¹⁶ and R¹⁸independently are halogen, hydrogen, hydroxyl, thiol, amino, alkylamino, alkoxy, or —NHR^(c) where R^(c) is as defined above, and at leastone of R¹⁶ and R¹⁸ is other than hydrogen. In certain embodiments, thecompounds have a chemical structure according to general formula (IV)where X¹ is oxygen and R¹⁹ and R²¹ independently are hydroxyl, thiol,oxygen, imino, iminium, alkyl imino, alkyl iminium, amino, or alkylamino, and at least one of R¹⁹ and R²¹ is other than hydrogen; inparticular embodiments, R¹⁸ is halogen, hydroxyl, thiol, amino, alkylamino, alkoxy, or —NHR^(c) where R^(c) is as defined above. In someembodiments, the compounds have a chemical structure according togeneral formula (VI) where R¹⁷ is halogen, hydroxyl, thiol, amino, alkylamino, or —NHR^(c) where R^(c) is as defined above, and R²⁰ is oxygen,sulfur, imino, iminium, alkyl iminium, or —NHR^(c) where R^(c) is asdefined above. In some embodiments, the compounds have a chemicalstructure according to general formula (III) where X¹ is oxygen and R⁶and R¹⁶ are —NHR^(c) or general formula (IV) where X¹ is oxygen and R¹⁶and R²¹ are —NHR^(c) where R^(c) is as defined above.

In particular embodiments, the compound has a chemical structureselected from

In some embodiments, a method for using the disclosed compounds todetect analytes in biological fluid includes combining a compound havinga structure according to any one of general formulas (III)-(XI) with abiological fluid to form a solution, exposing the solution to a lightsource, and detecting the analyte by detecting fluorescence from thecompound. In certain embodiments, the light source has a wavelength inthe range of 190 nm to 850 nm. Detecting fluorescence from the compoundmay include detecting fluorescence at a wavelength corresponding to anemission spectrum maximum of the compound. In certain embodiments, thecompound has an emission spectrum maximum at a wavelength greater thanor equal to 700 nm, or greater than or equal to 750 nm. In particularembodiments, the analyte is quantitated by measuring an amount offluorescence from the compound at a wavelength corresponding to anemission spectrum maximum of the compound.

In some embodiments, the analyte is detected in a biological fluidcomprising blood or urine. In certain embodiments, the analyte iscysteine, homocysteine, glutathione,succinyl-5-amino-4-imidazolecarboxamide riboside, succinyladenosine, ora combination thereof. In some embodiments, when detecting glutathione,succinyl-5-amino-4-imidazolecarboxamide riboside, succinyladenosine, ora combination thereof, the compound may have a chemical structureaccording to general formula (VI) where R¹, R⁴, R⁵ and R⁸ independentlyare hydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino,substituted amino, alkoxy, or halogen; and R¹⁹-R²² independently arehydrogen, hydroxyl, thiol, halogen, oxygen, imino, iminium, alkyl imino,alkyl iminium, amino, alkyl amino, or —NHR^(c) where R^(c) is as definedabove. For example, when the analyte is glutathione, R¹⁷ may behydroxyl, amino, or alkyl amino, and R²⁰ may be oxygen, hydroxyl, amino,alkyl amino, imino, or alkyl iminium. In certain embodiments, when theanalyte is glutathione, the compound may be

When the analyte is succinyl-5-amino-4-imidazolecarboxamide riboside,succinyladenosine, or a combination thereof, at least one of R¹⁷ and R²⁰may be —NHR^(c). In such embodiments, R¹³ typically is —COOR¹⁴ where R¹⁴is hydrogen or lower alkyl, and the linker is at R⁹, R¹⁰, R¹¹, or R¹².In some embodiments, the analyte issuccinyl-5-amino-4-imidazolecarboxamide riboside, succinyladenosine, ora combination thereof, and the compound comprises at least onefluorophore according to general formula (III) where X¹ is oxygen and R⁶and R¹⁶ are —NHR^(c) or general formula (IV) where X¹ is oxygen and R¹⁶and R²¹ are —NHR^(c).

Kits for detecting an analyte include at least one of the disclosedcompounds, wherein the compound when combined with a sample (e.g., abiological fluid) including the analyte will undergo a change in color,absorbance spectrum, emission spectrum, or a combination thereofcompared to the compound in a sample that does not include the analyte.In some embodiments, the kit further includes a buffer solution suitablefor effecting a change in the compound's absorbance and/or emissionspectrum when the compound and buffer are combined with a sampleincluding the analyte. The kit also may include color comparison chartfor evaluating a color change produced by a reaction between thecompound and the analyte. In certain embodiments, the kit includes aplurality of disposable containers in which a reaction between thecompound and the analyte can be performed. An amount of the compoundeffective to undergo a detectable change in the color, absorbancespectrum, the emission spectrum, or a combination thereof when reactedwith the analyte may be premeasured into the plurality of disposablecontainers.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 includes general chemical structures of type [c] annulatedxanthenes with substituents at carbon-1 or carbon-3 of the annulatedring, where R is —OH or —N(CH₃)₂, R′ is oxygen or —N(CH₃)₂ ⁺, and R″ ishydrogen or lower alkyl.

FIG. 2 is a reaction scheme illustrating the synthesis of someembodiments of the disclosed dyes having a chemical structure accordingto general formula IV.

FIG. 3 is a reaction scheme illustrating the synthesis of someembodiments of the disclosed dyes having a semi-naphthofluoresceinchemical structure according to general formula III.

FIG. 4 is a reaction scheme illustrating the synthesis of someembodiments of the disclosed dyes having a rhodol or rhodamine chemicalstructure according to general formula III.

FIG. 5 is a reaction scheme illustrating the synthesis of one embodimentof the disclosed dyes having a chemical structure according to generalformula IV.

FIG. 6 is a reaction scheme illustrating the synthesis of someembodiments of the disclosed dyes having chemical structures accordingto general formulas VI and VIII-XI.

FIG. 7 is a reaction scheme illustrating the synthesis of someembodiments of the disclosed dyes having chemical structures accordingto general formulas V and VII.

FIG. 8 is a reaction scheme illustrating the synthesis of someembodiments of the disclosed dyes having chemical structures accordingto general formula I where X¹ is Si(CH₃)₂, Ge(CH₃)₂, or Sn(CH₃)₂.

FIG. 9 is a reaction scheme illustrating the synthesis of someembodiments of the disclosed dyes having chemical structures accordingto general formula I where X¹ is S or Se.

FIG. 10 is a reaction scheme illustrating the synthesis of someembodiments of the disclosed dyes having bis-boronic acid chemicalstructures according to general formula III.

FIG. 11 is a reaction scheme illustrating the synthesis of someembodiments of the disclosed dyes having bis-boronic acid chemicalstructures according to general formula IV.

FIG. 12 illustrates the molecular interactions of one embodiment of thedisclosed dyes, compound 24, with glutathione.

FIG. 13 illustrates the molecular interactions of two embodiments of thedisclosed dyes, compounds 43 and 44, with S-Ado (left) and SAICAr(right).

FIG. 14 is an x-ray structure of one embodiment of the disclosed dyes,compound 14a. FIG. 15 is a series of absorbance spectra of twoembodiments of the disclosed dyes, compounds 7 and 21, in their dianionand monanion states.

FIG. 16 is a series of absorbance spectra of two embodiments of thedisclosed dyes, compounds 8 and 22, in their neutral and anionic forms.

FIG. 17 is a series of absorbance spectra of two embodiments of thedisclosed dyes, compounds 15a and 20a, in their neutral and anionicforms.

FIG. 18 is a series of absorbance spectra of one embodiment of thedisclosed dyes, compound 15b, in its phenolic and phenoxide forms.

FIG. 19 is a series of absorbance spectra of one embodiment of thedisclosed dyes, compound 15d, in its phenolic and phenoxide forms.

FIG. 20 is a series of absorbance spectra of two embodiments of thedisclosed dyes, compounds 15g and 20b, in their cationic forms.

FIG. 21 is a series of pH titration curves of one embodiment of thedisclosed dyes, compound 15e.

FIG. 22 is a series of spectra of one embodiment of the disclosed dyes,compound 15e, in buffer and in 5% whole blood in buffer.

FIG. 23A is an absorption spectrum of one embodiment of the discloseddyes, compound 15g, in 10% whole blood.

FIG. 23B is a series of emission spectra of one embodiment of thedisclosed dyes compound 15g, in 10% and 90% whole blood.

FIG. 24 is a series of absorption spectra showing the effect ofionization on the absorption maxima of several embodiments of thedisclosed dyes.

FIG. 25 shows the intramolecular hydrogen-bond network of one embodimentof the disclosed dyes, compound 8.

FIG. 26 is a molecular simulation showing that hydrogen bonding enhancescoplanarity of the fused ring system for one embodiment of the discloseddyes, compound 8, which is diminished upon deprotonation.

FIGS. 27A-F are absorption and fluorescence spectra of two embodimentsof rhodamine bis-boronic acids, compounds 51 and 53, and theirrespective precursors, compounds 50 and 52. Spectra were obtained inDMSO:buffer 1:9 (FIGS. 27A-C) and DMSO:buffer 1:1 (FIGS. 27D-F).

FIG. 28 is a series of DMSO titration curves for two embodiments ofrhodamine bis-boronic acids compounds 51 and 53, and their respectiveprecursors, compounds 50 and 52; final pH 7.4 phosphate bufferconcentration was 12.5 mM.

FIGS. 29A-D are absorbance and fluorescence spectra of one embodiment ofa rhodamine bis-boronic acid, compound 51, in response to varioussaccharides. FIG. 29A is absorption and emission spectra of the compound(3.75 μM) in response to 10 mM sugars in DMSO:buffer 1:9; FIG. 29B isfluorescence emission (ex. 580 nm/em. 660 nm) as a function of sugarconcentration in DMSO:buffer 1:9; FIG. 29C is absorption and emissionspectra of the compound (3.75 μM) in response to 10 mM sugars inDMSO:buffer 1:1; FIG. 29D is fluorescence emission (ex. 580 nm/em. 640nm) as a function of sugar concentration in DMSO:buffer 1:1. The finalpH 7.4 phosphate buffer concentration was 12.5 mM.

FIGS. 30A-D are absorbance and fluorescence spectra of one embodiment ofa rhodamine bis-boronic acid, compound 53, in response to varioussaccharides. FIG. 30A is absorption (630 nm) spectra of the compound(7.5 μM) as a function of sugar concentration in DMSO:buffer 1:9; FIG.30B is fluorescence (ex. 630/em. 690 nm) spectra of the compound (7.5μM) as a function of sugar concentration in DMSO:buffer 1:9; FIG. 30C isabsorption (640 nm) spectra of the compound (7.5 μM) as a function ofsugar concentration in DMSO:buffer 1:1; FIG. 30D is fluorescence (ex.640/em. 700 nm) spectra of the compound (7.5 μM) as a function of sugarconcentration in DMSO:buffer 1:1; the final pH 7.4 phosphate bufferconcentration was 12.5 mM.

FIG. 31 shows the energy-minimized structure of one embodiment of acomplex formed between a ribose and one embodiment of a rhodaminebis-boronic acid compound, compound 51.

FIG. 32 shows the energy-minimized structure of one embodiment of acomplex formed between two molecules of fructose and one embodiment of arhodamine bis-boronic acid compound, compound 53.

DETAILED DESCRIPTION

Disclosed herein are embodiments of novel near-infrared (NIR) dyes basedon xanthene architectures. Xanthene dyes such as fluorescein andrhodamine derivatives are commonly used fluorescent dyes due to theirbright fluorescence and compatibility with common laser lineexcitations. Fluorescein emission generally falls in the green or yellowspectral region while some rhodamines exhibit red emission (>600 nm).

Embodiments of the disclosed NIR dyes form a unique series ofxanthene-based regioisomeric naphthofluorone dyes exhibiting acombination of desirable characteristics, including (i) relatively lowmolecular weight, (ii) aqueous solubility, and/or (iii) dual excitationand emission from their fluorescent neutral and anionic forms.Systematic changes in the regiochemistry of benzannulation and theionizable moieties afford (iv) tunable deep-red to NIR emission and (v)enhanced Stokes shifts.

I. Terms and Definitions

The following explanations of terms and abbreviations are provided tobetter describe the present disclosure and to guide those of ordinaryskill in the art in the practice of the present disclosure. As usedherein, “comprising” means “including” and the singular forms “a” or“an” or “the” include plural references unless the context clearlydictates otherwise. The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features of thedisclosure are apparent from the following detailed description and theclaims.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, percentages, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Unless otherwise indicated,non-numerical properties such as amorphous, crystalline, homogeneous,and so forth as used in the specification or claims are to be understoodas being modified by the term “substantially,” meaning to a great extentor degree. Accordingly, unless otherwise indicated, implicitly orexplicitly, the numerical parameters and/or non-numerical properties setforth are approximations that may depend on the desired propertiessought, limits of detection under standard test conditions/methods,limitations of the processing method, and/or the nature of the parameteror property. When directly and explicitly distinguishing embodimentsfrom discussed prior art, the embodiment numbers are not approximatesunless the word “about” is recited.

Definitions of common terms in chemistry may be found in Richard J.Lewis, Sr. (ed.), Hawley's Condensed Chemical Dictionary, published byJohn Wiley & Sons, Inc., 1997 (ISBN 0-471-29205-2). Definitions ofcommon terms in molecular biology may be found in Benjamin Lewin, GenesVII, published by Oxford University Press, 2000 (ISBN 019879276X);Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by Wiley, John & Sons, Inc., 1995 (ISBN0471186341); and other similar references.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Absorbance is the retention by a compound or substance of certainwavelengths of radiation incident upon it; a measure of the amount oflight at a particular wavelength absorbed as the light passes through acompound or substance, or through a solution of a compound or substance.

Alkyl refers to a hydrocarbon group having a saturated carbon chain. Thechain may be cyclic, branched or unbranched. The term lower alkyl meansthe chain includes 1-10 carbon atoms.

An analogue or derivative is a molecule that differs in chemicalstructure from a parent compound, for example a homologue (differing byan increment in the chemical structure, such as a difference in thelength of an alkyl chain), a molecular fragment, a structure thatdiffers by one or more functional groups, a change in ionization.Structural analogues are often found using quantitative structureactivity relationships (QSAR), with techniques such as those disclosedin Remington (The Science and Practice of Pharmacology, 19th Edition(1995), chapter 28).

Annelation/annulation is a chemical reaction in which one cyclic or ringstructure is added to another to form a polycyclic, or annulated,compound. Annulation can be categorized as type [a], type [b], or type[c], depending on the position of the newly added ring. Type [a] refersto “down annulation,” type [b] refers to “across annulation,” and type[c] refers to “up annulation,” as shown below:

As used herein, the term “annulated” refers to having or consisting ofrings or ringlike segments. The term “benzannulated” refers toderivatives of cyclic compounds (usually aromatic), which are fused to abenzene ring. Examples of benzannulated compounds include, inter alia,benzopyrenes, quinolines, naphthoquinones, naphthofluoresceins,rhodamines, and xanthenes.

Aromatic or aryl compounds typically are unsaturated, cyclichydrocarbons having alternate single and double bonds. Benzene, a6-carbon ring containing three double bonds, is a typical aromaticcompound.

A bathochromic shift is a change of spectral band position in theabsorption, reflectance, transmittance, or emission spectrum of amolecule to a longer wavelength, or lower frequency. A bathochromicshift commonly is referred to as a “red shift.” Bathochromic shifts canoccur in the spectra of a series of structurally related molecules withdifferent substitutions and/or substitution patterns. A change inenvironment, e.g., solvent polarity, also can produce a bathochromicshift.

Coumarin: A benzopyrone having the following general structure:

As used herein, “substituted coumarin” refers to a coumarin includingone or more substituents. Suitable substituents may include substitutedor unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy,lower alkyl carbonyl, substituted or unsubstituted amino, halo, andhydroxyl groups. In some embodiments, the coumarin is substituted at the3- and/or 7-position with, for example, a lower alkyl, lower alkylcarbonyl, lower alkoxy, amino or substituted amino group.

Emission or an emission signal refers to the light of a particularwavelength generated from a source. In particular examples, an emissionsignal is emitted from a fluorophore after the fluorophore absorbs lightat its excitation wavelength(s).

Excitation or an excitation signal refers to the light of a particularwavelength necessary and/or sufficient to excite an electron transitionto a higher energy level. In particular examples, an excitation signalis the light of a particular wavelength necessary and/or sufficient toexcite a fluorophore to a state such that the fluorophore will emit adifferent (such as a longer) wavelength of light than the wavelength oflight from the excitation signal.

Fluorescence is the emission of visible radiation by an atom or moleculepassing from a higher to a lower electronic state, wherein the timeinterval between absorption and emission of energy is 10⁻⁸ to 10⁻³second. As used herein, fluorescence occurs when the atom or moleculeabsorbs energy from an excitation source (e.g., a lamp producing lightwithin a wavelength range of 190-850 nm) and then emits the energy asvisible and/or near-infrared radiation.

A fluorogen is a compound capable of fluorescence.

A fluorophore is the functional group, or portion, of a molecule thatcauses the molecule to fluoresce when exposed to an excitation source.The term “fluorophore” also is used to refer to fluorescent compoundsused as dyes to mark proteins with a fluorescent label.

A functional group is a specific group of atoms within a molecule thatis responsible for the characteristic chemical reactions of themolecule. Exemplary functional groups include, without limitation,alkane, alkene, alkyne, arene, halo (fluoro, chloro, bromo, iodo),epoxide, hydroxyl, carbonyl (ketone), aldehyde, carbonate ester,carboxylate, ether, ester, peroxy, hydroperoxy, carboxamide, amine(primary, secondary, tertiary), ammonium, imide, azide, cyanate,isocyanate, thiocyanate, nitrate, nitrite, nitrile, nitroalkane,nitroso, pyridyl, phosphate, sulfonyl, sulfide, thiol (sulfhydryl),disulfide.

GSH: Glutathione.

Heteroaryl compounds are aromatic compounds having at least oneheteroatom, i.e., one or more carbon atoms in the ring has been replacedwith an atom having at least one lone pair of electrons, typicallynitrogen, oxygen, or sulfur.

Imino refers to a functional group having the formula ═NH.

Iminium refers to a protonated or substituted imine, e.g., ═NH₂ ⁺ or(═NR_(A)R_(B))⁺ where R_(A) and R_(B) represent alkyl or substitutedalkyl groups. As used herein, (═NR_(A)R_(B))⁺ is referred to as an alkyliminium group.

MIP: Molecule-imprinted polymer.

Near infrared (NIR) is a region of the electromagnetic spectrum betweenthe visible region and the infrared region. There is no set definitionfor the boundaries of the near-infrared region, but definitions includethe wavelength ranges from 650-2500 nm, 750-2500 nm, 780-2500 nm,800-2500 nm, 700-1400 nm, or 780-3000 nm. As used herein, NIR typicallyrefers to the wavelength region of 700-1400 nm.

A rhodol is a structural hybrid of fluorescein and a rhodamine.Rhodamines are a family of related fluorone dyes. The structures offluorescein, a rhodamine, and two rhodol analogues are shown below.

SNAFL: Seminaphthofluorescein

SNAFR: Seminaphthofluorone

SNARF: Seminaphthorhodafluor

Stokes shift refers to the difference (in wavelength or frequency units)between absorbance spectrum maximum and the emission spectrum maximum ofthe same electronic transition. Typically, the wavelength of maximumfluorescence emission is longer than that of the exciting radiation,i.e., the wavelength of maximum absorbance.

Xanthene is an organic heterocyclic compound with the formula C₁₃H₁₀O.

Xanthene derivatives are referred to as xanthenes, and includefluorescein, rhodamine, and derivatives thereof.II. Overview Of Representative Embodiments

Embodiments of compounds having emission spectrum maxima in thenear-infrared region (near-infrared (NIR) dyes) and that selectivelydetect analytes in buffered solutions and/or biological media aredisclosed. Some embodiments of the disclosed compounds exhibit largebathochromic shifts and/or enhanced Stokes shifts compared to currentlyavailable NIR dyes.

Some embodiments of the disclosed compounds have a structure accordingto general formulas (III)-(XI) as described herein. In some embodiments,X¹ is oxygen. In any or all of the above embodiments, R¹³ may be —COO—and form a lactone ring. In any or all of the above embodiments, thecompound may comprise at least one halogen atom positioned adjacent toan ionizable moiety.

In any or all of the above embodiments, the compound may have anemission spectrum maximum at a wavelength greater than or equal to 700nm, such as greater than or equal to 750 nm. In any or all of the aboveembodiments, the compound may have a Stokes shift greater than or equalto 80 nm, such as greater than or equal to 100 nm.

In some embodiments, the compound has a chemical structure according tostructure (III) where R⁶ is amino or alkyl amino, and at least one ofR¹⁶ or R¹⁸ is hydroxyl, amino, or alkyl amino. In other embodiments, thecompound has a chemical structure according to structure (III), whereR¹, R², R⁵, R⁷, and R⁸ are hydrogen or halogen; R⁶ is oxygen, imino,iminium, lower alkyl iminium, or —NHR^(c), and R⁹-R¹² independently arehydrogen, amino, lower alkyl, carboxyl, or —SO₃H. In still otherembodiments, the compound has a chemical structure according tostructure (III) where R¹, R², R⁵, R⁷-R¹², and R¹⁵-R¹⁷ are hydrogen, R⁶is iminium, R¹³ is —COOR¹⁴ where R¹⁴ is hydrogen or lower alkyl, and R¹⁸is amino.

In some embodiments, the compound has a chemical structure according tostructure (IV), (IX), or (X) where X¹ is oxygen and R¹⁸ is halogen,hydroxyl, thiol, amino, alkyl amino, or alkoxy. In some embodiments, thecompound has a chemical structure according to structure (IV) where X¹is oxygen and R¹⁹ is hydroxyl, thiol, oxygen, imino, iminium, alkylimino, alkyl iminium, amino, or alkyl amino; in certain embodiments, R¹⁸is halogen, hydroxyl, thiol, amino, alkyl amino, or alkoxy.

In some embodiments, the compound has a chemical structure according tostructure (VI) where R¹⁷ is halogen, hydroxyl, thiol, amino, alkylamino, or —NHR^(c), and R²⁰ is oxygen, sulfur, imino, iminium, alkyliminium, or —NHR^(c).

Embodiments of a method for selectively detecting an analyte in abiological fluid include combining a compound according to any or all ofthe above embodiments with a biological fluid to form a solution,exposing the solution to a light source, and detecting the analyte bydetecting fluorescence from the compound. In some embodiments, the lightsource has a wavelength in the range of 190 nm to 850 nm. In any or allof the above embodiments, the biological fluid may comprise blood orurine.

In any or all of the above embodiments, detecting fluorescence from thecompound may include detecting fluorescence at a wavelengthcorresponding to an emission spectrum maximum of the compound. In someembodiments, the compound has an emission spectrum maximum at awavelength greater than or equal to 700 nm, such as greater than orequal to 750 nm. In some embodiments, the analyte is quantitated bymeasuring an amount of fluorescence from the compound at a wavelengthcorresponding to an emission spectrum maximum of the compound.

In any or all of the above embodiments, the analyte may be cysteine,homocysteine, glutathione, succinyl-5-amino-4-imidazolecarboxamideriboside, succinyladenosine, or a combination thereof. In someembodiments, the compound has a chemical structure according to generalstructure (VI) where R¹, R⁴, R⁵ and R⁸ independently are hydrogen,hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, substituted amino,alkoxy, or halogen; and R¹⁹-R²² independently are hydrogen, hydroxyl,thiol, halogen, oxygen, imino, iminium, alkyl imino, alkyl iminium,amino, alkyl amino, or —NHR^(c).

In some embodiments, the analyte is glutathione, and the compound has astructure according to general structure (VI) as described above whereR¹⁷ is hydroxyl, amino, or alkyl amino, and R²⁰ is oxygen, hydroxyl,amino, alkyl amino, imino, or alkyl iminium. In one embodiment, thecompound is

In some embodiments, the analyte issuccinyl-5-amino-4-imidazole-carboxamide riboside, succinyladenosine, ora combination thereof, and the compound has a structure according togeneral structure (VI) as described above where at least one of R¹⁷ andR²⁰ is —NHR^(c), and R¹³ is —COOR¹⁴ where R¹⁴ is hydrogen or loweralkyl. In some embodiments, the analyte issuccinyl-5-amino-4-imidazolecarboxamide riboside, succinyladenosine, ora combination thereof, and the compound comprises at least onefluorophore according to general formula (III) where X¹ is oxygen and R⁶and R¹⁶ are —NHR^(c) or general formula (IV) where X¹ is oxygen and R¹⁶and R²¹ are —NHR^(c).

Embodiments of a kit for detecting an analyte include at least onecompound according to any or all of the above embodiments, wherein thecompound when combined with a sample comprising the analyte will undergoa change in its absorbance spectrum and/or emission spectrum compared tothe compound in a sample that does not comprise the analyte. In someembodiments, the sample is a biological fluid. In any or all of theabove embodiments, the kit may include at least one buffer solution inwhich the compound when combined with a sample comprising the analytewill undergo a change in its absorbance spectrum and/or emissionspectrum compared to the compound combined with the buffer solution anda sample that does not comprise the analyte. In any or all of the aboveembodiments, the analyte may be glutathione, cysteine, homocysteine,succinyl-5-amino-4-imidazolecarboxamide riboside, succinyladenosine, ora combination of succinyl-5-amino-4-imidazolecarboxamide riboside andsuccinyladenosine. In some embodiments, the compound in the kit has achemical structure according to general structure (VI) as describedabove. In some embodiments, the compound in the kit has a chemicalstructure according to general structure (III) as described above whereX¹ is oxygen and R⁶ and R¹⁶ are —NHR^(c) or general structure (IV) whereX¹ is oxygen and R¹⁶ and R²¹ are —NHR^(c).

In any or all of the above embodiments, the kit may include a colorcomparison chart for evaluating a color change produced by a reactionbetween the compound and the analyte. In any or all of the aboveembodiments, the kit may include a plurality of disposable containers inwhich a reaction between the compound and the analyte can be performed.In some embodiments, an amount of the compound effective to undergo adetectable change in the absorbance spectrum, the emission spectrum, orboth when reacted with the analyte is premeasured into the plurality ofdisposable containers.

III. Near-Infrared Dyes

Embodiments of the disclosed near-infrared (NIR) dyes are based on anannulated xanthene architecture. Some embodiments of the disclosed NIRdyes exhibit significant bathochromic shifts and enhanced Stokes shiftscompared to structurally related analogues. In some embodiments, the NIRdyes are benzannulated xanthenes with single or double annulation. Theannulation may be type [a]—down, type [b]—across, or type [c]—up. Atleast some embodiments of the singly and doubly annulated NIR dyes havesubstantially red-shifted absorbance and emission spectra compared tocommercially available NIR dyes. In some embodiments, the spectra arered shifted by at least 100 nm. In particular, some embodiments of thedisclosed NIR dyes have an emission maximum greater than 700 nm. Incertain embodiments, emission maxima are well into the near-infraredregion, e.g., greater than 700 nm, greater than 750 nm, and even greaterthan 780 nm. In comparison, some known annulated xanthenes have emissionmaxima near 560 nm (fully benzannulated dibenzofluorescein), 630 nm(SNAFLs), 650 nm (SNARFs), 670 nm (naphthofluorescein), or 650-675 nm(dibenzorhodamines). In some embodiments, the absorption maxima aregreater than 600 nm, greater than 650 nm, greater than 675 nm, and evengreater than 800 nm.

Some embodiments of the NIR dyes also exhibit an enhanced Stokes shiftcompared to known long-wavelength xanthenes. In certain embodiments, theStokes shift (difference between absorbance spectrum maximum and theemission spectrum maximum of the same electronic transition) is greaterthan 50 nm, greater than 80 nm, greater than 100 nm, or greater than 150nm, such as 50-200 nm, 50-150 nm, 80-150 nm, 90-170 nm, 100-150 nm, or100-200 nm. A large Stokes shift is advantageous since it facilitatesuse of relatively broad-band light sources, such as light-emittingdiodes, with minimal scattered light interference from the light sourceat the emission wavelength(s) measured.

A. Structures

In some embodiments, the NIR dyes have a structure according to generalformula I.

In general formula I, each bond depicted as “

” is a single or double bond as needed to satisfy valence requirements;X¹ is O, S, Se, Si(CH₃)₂, Ge(CH₃)₂, Sn(CH ₃)₂, CH₂, C(CH₃)₂ or NH. R¹,R², and R⁴ independently are hydrogen, hydroxyl, oxygen, thiol, loweralkyl, carboxyalkyl, amino, substituted amino, alkoxy, halogen, or—NHR^(c) where R^(c) is

R⁵, R⁷, and R⁸ independently are hydrogen, hydroxyl, thiol, lower alkyl,carboxyalkyl, amino, substituted amino, alkoxy, or halogen; R³ and R⁶independently are hydrogen, hydroxyl, halogen, oxygen, sulfur, thiol,amino, alkyl amino, imino, iminium, alkyl imino, alkyl iminium,cycloalkyl imino, or —NHR^(c) where R^(c) is as defined above; and atleast one of R¹ and R², R² and R³, R³ and R⁴, R⁵ and R⁶, R⁶ and R⁷,and/or R⁷ and R⁸ together form a substituted or unsubstituted cycloalkylor aryl. R⁹-R¹² independently are hydrogen, alkyl, acyl, carboxyl,nitro, amino, alkyl amino, or —SO₃H. R¹³ is hydrogen, hydroxyl, loweralkyl, lower alkoxy, —SO₃H or —COOR¹⁴ where R¹⁴ is hydrogen or loweralkyl and the bond depicted as “

” in ring B is a double bond, or R¹³ is one or more atoms forming a ringsystem with rings B and D and the bond depicted as “

” in ring B is a single bond. In some embodiments, R¹³ is —COO— andforms a lactone ring as shown in general formula II.

In some embodiments, R³ and R⁴ and/or R⁵ and R⁶ together form an aryl orsubstituted aryl ring as shown in general formulas III and IV

where each bond depicted as “

” is a single or double bond as needed to satisfy valence requirements.R¹-R¹⁴ and X¹ are as defined for general formulas (I) and (II). R¹⁵R²²independently are hydrogen, halogen, hydroxyl, oxygen, thiol, amino,alkyl amino, alkoxy, such as lower alkoxy, sulfur, imino, iminium, alkylimino, alkyl iminium, or —NHR^(c) where R^(c) is as defined above. R¹⁹,R²⁰, R²¹, and R²² independently are hydrogen, hydroxyl, thiol, halogen,oxygen, amino, alkyl amino, or —NHR^(c) where R^(c) is as defined above.In certain embodiments, X¹ is oxygen, R¹, R², R⁵, R⁷ and R⁸independently are hydrogen or halogen; R⁶ is oxygen, imino, iminium,lower alkyl iminium, or —NHR^(c) where R^(c) is as defined above; R⁹-R¹²independently are hydrogen, amino, lower alkyl, carboxyl, or —SO₃H; R¹⁵,R¹⁷, R²⁰, and R²² are hydrogen; R¹³ is hydrogen, lower alkyl, loweralkoxy, —SO₃H, —COOR¹⁴ where R¹⁴ is hydrogen or lower alkyl and the bonddepicted as “

” in ring B is a double bond, or R¹³ is one or more atoms forming a ringsystem with rings B and D and the bond depicted as “

” in ring B is a single bond; R¹⁶ and R¹⁸ independently are hydrogen,hydroxyl, oxygen, lower alkoxy, amino, alkyl amino, or —NHR^(c) whereR^(c) is as defined above, and at least one of R¹⁶ and R¹⁸ is other thanhydrogen; R¹⁹ and R²¹ independently are oxygen, imino, iminium, loweralkyl iminium, or —NHR^(c) where R^(c) is as defined above, and at leastone of R¹⁹ and R²¹ is other than hydrogen. In particular embodiments, X¹is oxygen, R¹, R², R⁵, R⁸, R⁹, R¹², R¹⁵, R¹⁷, R²⁰, and R²² are hydrogen;R⁶ is oxygen, imino, iminium, lower alkyl iminium, or —NHR^(c) whereR^(c) is as defined above; R⁷ is hydrogen or halogen; R¹⁰ and R¹¹independently are hydrogen, amino, lower alkyl, carboxyl, or —SO₃H; R¹³is hydrogen, lower alkyl, lower alkoxy, —SO₃H, or —COOR¹⁴ where R¹⁴ ishydrogen or lower alkyl and the bond depicted as “

” in ring B is a double bond, or R¹³ is one or more atoms forming a ringsystem with rings B and D and the bond depicted as “

” in ring B is a single bond; R¹⁶ and R¹⁸ independently are hydrogen,hydroxyl, oxygen, lower alkoxy, amino, alkyl amino, or —NHR^(c) whereR^(c) is as defined above, and at least one of R¹⁶ and R¹⁸ is other thanhydrogen; R¹⁹ is hydrogen, hydroxyl, oxygen, imino, iminium, or loweralkyl iminium; R²¹ is hydrogen, oxygen, or —NHR^(c) where R^(c) is asdefined above, and at least one of R¹⁹ and R²¹ is other than hydrogen.In certain embodiments, if X¹ is oxygen in structure III, then R⁶ isother than oxygen or alkyl amino, or R¹³ is other than one or more atomsforming a ring system with rings B and D, or R¹⁶ is other than hydroxylor hydrogen, or R¹⁸ is other than hydroxyl or hydrogen, or at least oneof R¹, R², R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², or R¹³ is other than hydrogen.In certain embodiments, if X¹ is oxygen, sulfur, CH₂, or NH in structureIV, then at least one of R¹, R², R⁷-R¹³, or R¹⁵-R²² is other thanhydrogen, hydroxyl, halogen, lower alkyl, amino, or thiol, or R¹³ is oneor more atoms forming a ring system with rings B and D and the bonddepicted as “

” in ring B is a single bond. Representative compounds according togeneral formulas III and IV are shown in Table 1.

TABLE 1 Cpd R⁶* R⁷ R¹⁰ R¹¹ R¹³ R¹⁶ R¹⁸ R¹⁹ R²¹  3a O H H H —OCH₃ H —OH ——  3b O H —CH₃ H —CH₃ H —OH — —  3c O H H —COOH H H —OH — —  3d O H H—NH₂ H H —OH — —  3e O H H H —SO₃H H —OH — —  3f O H —SO₃H H —SO₃H H —OH— —  3g O F H H H H —OH — —  3h ═NH H —SO₃H H —SO₃H H —NH₂ — —  3i═N(CH₃)₂ ⁺ H H H H H —OH — —  3j ═N(CH₃)₂ ⁺ H H H H H —N(CH₃)₂ — —  3k═N(CH₃)₂ ⁺ H —SO₃H H —SO₃H H —N(CH₃)₂ — —  4a — H —CH₃ H —CH₃ H —OH ═O H 4b — H —SO₃H H —SO₃H H —OH ═O H  4c — H —CH₃ H —CH₃ H —NH₂ ═NH H  4d —H —CH₃ H —CH₃ H —N(CH₃)₂ ═N(CH₃)₂ ⁺ H  4e — H H —COOH H H —OH O H  4f —H H —COOH H H —N(CH₃)₂ ═N(CH₃)₂ ⁺ H  7 — H H H —COO^(†) H —OH —OH H  7a— H H H —COO⁻ H —OH —OH H  7b — H H H —COO⁻ H —O− —OH H  8 — H H H—COOCH₃ H —OH ═O H  8a — H H H —COOCH₃ H —O⁻ ═O H  9 — H H H —COOCH₃ H—OCH₃ ═O H 10 — H —CH₃ H —CH₃ H —OH ═O H 11 — H H —SO₃H —SO₃H H —OH ═O H14a —OH H H H —COO−^(†) H —OH — — 14b —NH₂ H H H —COO−^(†) H —OH — — 14c—NH₂ H H H —COO−^(†) H —OCH₃ — — 14d —N(CH₃)₂ H H H —COO−^(†) H —OH — —14e —OCH₃ H H H —COO−^(†) H —OH — — 14f —OH H H H —COO−^(†) H —NH₂ — —14g —OH H H H —COO−^(†) H —N(CH₃)₂ — — 14h —NH₂ H H H —COO−^(†) H —NH₂ —— 15a ═O H H H —COOCH₃ H —OH — — 15b ═NH₂ ⁺ H H H —COOCH₃ H —OH — — 15c═NH₂ ⁺ H H H —COOCH₃ H —OCH₃ — — 15d ═N(CH₃)₂ ⁺ H H H —COOCH₃ H —OH — —15e ═O H H H —COOCH₃ H —NH₂ — — 15f ═O H H H —COOCH₃ H —N(CH₃)₂ — — 15g═NH₂ ⁺ H H H —COOCH₃ H —NH₂ — — 15h ═O H H H —COOCH₃ H —O⁻ — — 16a ═O HH H —COOCH₃ H —OCH₃ — — 16b ═N(CH₃)₂ ⁺ H H H —COOCH₃ H —OCH₃ — — 19a —OHH H H —COO−^(†) —OH H — — 20a ═O H H H —COOCH₃ —OH H — — 20b ═⁺NH₂ H H H—COOCH₃ —NH₂ H — — 20c ═O H H H —COOCH₃ —O⁻ H — — 20d ═O H H H —COOH —OHH — — 21a ═O H H H —COOH —OH H — — 21b ═O H H H —COO− —O− H — — 22 — H HH —COOCH₃ —OH H H ═O 22a — H H H —COOH —OH H H ═O 40 — H H H —COOCH₃ H—OH ═O H 41 —N(CH₃)₂ H H H —COO−^(†) H —N(CH₃)₂ — — 42 ═N(CH₃)₂ ⁺ H H H—COOCH₃ H —N(CH₃)₂ — — 50 ═NH₂ ⁺ H H H —COO⁻ ═NH₂ ⁺ H — — 51 —NHR^(c+) HH H —COO⁻ —NHR^(c+) H — — 52 — H H H —COO⁻ ═NH₂ ⁺ H H ═NH₂ ⁺ 53 — H H H—COO⁻ —NHR^(c+) H H —NHR^(c+) *All R substituents not included in Table1 are hydrogen and X¹ is oxygen. ^(†)R¹¹ forms a lactone ring.

In some embodiments, R² and R³ and/or R⁶ and R⁷ together form an aryl orsubstituted aryl ring as shown in general formulas V and VI

where each bond depicted as “

” is a single or double bond as needed to satisfy valence requirements,and R¹-R²² and X¹ are defined as above. In certain embodiments, X¹ isoxygen, R¹, R⁴, R⁵, R⁷ and R⁸ independently are hydrogen or halogen; R⁶is oxygen, imino, iminium, lower alkyl iminium, or —NHR^(c) where R^(c)is as defined above; R⁹-R¹² independently are hydrogen, amino, loweralkyl, carboxyl, or —SO₃H; R¹⁵, R¹⁷, R¹⁹, and R²² are hydrogen; R¹³ ishydrogen, lower alkyl, lower alkoxy, —SO₃H, —COOR¹⁴ where R¹⁴ ishydrogen or lower alkyl and the bond depicted as “

” in ring B is a double bond, or R¹³ is one or more atoms forming a ringsystem with rings B and D and the bond depicted as “

” in ring B is a single bond; at least one of R¹⁶, R¹⁷ and R¹⁸ ishydroxyl, oxygen, amino, alkyl amino, or —NHR^(c) where R^(c) is asdefined above; and at least one of R¹⁹, R²⁰ and R²¹ is oxygen, imino,iminium, lower alkyl iminium, or —NHR^(c) where R^(c) is as definedabove. In particular embodiments, X¹ is oxygen, R¹, R⁴, R⁵, R⁸, R⁹, R¹²,R¹⁵, R¹⁶, R¹⁸, R¹⁹, R²¹, and R²² are hydrogen; R⁶ is oxygen, imino,iminium, lower alkyl iminium, or —NHR^(c) where R^(c) is as definedabove; R⁷ is hydrogen or halogen; R¹⁰ and R¹¹ independently arehydrogen, amino, lower alkyl, carboxyl, or —SO₃H; R¹³ is hydrogen, loweralkyl, lower alkoxy, —SO₃H, or —COOR¹⁴ where R¹⁴ is hydrogen or loweralkyl; R¹⁷ is hydroxyl, oxygen, amino, alkyl amino, or —NHR^(c) whereR^(c) is as defined above; and R²⁰ is oxygen, imino, iminium, loweralkyl iminium, or —NHR^(c) where R^(c) is as defined above. In certainembodiments, if X¹ is oxygen and R¹⁵ or R¹⁷ is hydroxyl in structure V,then R⁶ is other than oxygen, amino, or alkyl amino, or at least one ofR¹, R⁸, or R¹⁸ is other than hydrogen, or R¹³ is one or more atomsforming a ring system with rings B and D and the bond depicted as “

” in ring B is a single bond. Representative compounds according togeneral formulas V and VI are shown in Table 2.

TABLE 2 Cpd R⁶* R⁷ R¹⁰ R¹¹ R¹³ R¹⁷ R²⁰  2a O H H H —OCH₃ —OH —  2b O H—CH₃ H —CH₃ —OH —  2c O H H —COOH H —OH —  2d O H H —NH₂ H —OH —  2e O HH H —SO₃H —OH —  2f O H —SO₃H H —SO₃H —OH —  2g O F H H H —OH —  2h ═NHH —SO₃H H —SO₃H —NH₂ —  2i ═N(CH₃)₂ ⁺ H H H H —OH —  2j ═N(CH₃)₂ ⁺ H H HH —N(CH₃)₂ —  2k ═N(CH₃)₂ ⁺ H —SO₃H H —SO₃H —N(CH₃)₂ —  5a — H —CH₃ H—CH₃ —OH ═O  5b — H —SO₃H H —SO₃H —OH ═O  5c — H —CH₃ H —CH₃ —NH₂ ═NH 5d — H —CH₃ H —CH₃ —N(CH₃)₂ ═N(CH₃)₂ ⁺  5e — H H —COOH H —OH ═O  5f — HH —COOH H —N(CH₃)₂ ═N(CH₃)₂ ⁺ 24 — H H H —CH(OH)O−† —OH —NH₂ 34a — H H—COOH —CH₃ —OH ═O 34b — H H H —COOH —OH ═O 34c — H H —COOH —CH₃ —OH═N(CH₃)₂ ⁺ 34d — H H H —COOH —OH ═N(CH₃)₂ ⁺ 43 — H H H —COO−† —OH ═NH 44— H H H —COO−† —NH₂ ═NH 45 — H H H —COOH —OH ═NH 46 — H H H —COOH —NH₂═NH *All R substituents not included in Table 2 are hydrogen and X¹ isoxygen. ^(†)R¹¹ forms a ring.

In some embodiments, R¹ and R² and/or R⁷ and R⁸ together form an aryl orsubstituted aryl ring as shown in general formulas VII and VIII

where each bond depicted as “

” is a single or double bond as needed to satisfy valence requirements.R³-R¹⁸ and X¹ in general formula VII are defined as above. R³-R⁵,R⁹-R²², and X¹ in general formula VIII are defined as above, and R⁶ ishydrogen, hydroxyl, oxygen, halogen, thiol, amino, alkyl amino, or—NHR^(c) where R^(c) is as defined above. In certain embodiments, X¹ isoxygen, R³, R⁴, R⁵ independently are hydrogen or halogen; R⁷ and R⁸, ifpresent, independently are hydrogen or halogen; R⁹-R¹² independently arehydrogen, amino, lower alkyl, carboxyl, or —SO₃H; R¹⁵, R¹⁷, R¹⁹, and R²²are hydrogen; R¹³ is hydrogen, lower alkyl, lower alkoxy, —SO₃H, —COOR¹⁴where R¹⁴ is hydrogen or lower alkyl and the bond depicted as “

” in ring B is a double bond, or R¹³ is one or more atoms forming a ringsystem with rings B and D and the bond depicted as “

” in ring B is a single bond; at least one of R¹⁶, R¹⁷, and R¹⁸ ishydroxyl, oxygen, amino, alkyl amino, or —NHR^(c) where R^(c) is asdefined above; at least one of R¹⁹, R²⁰ and R²¹ is oxygen, imino,iminium, lower alkyl iminium, or —NHR^(c) where R^(c) is as definedabove; in general formula VII, R⁶ is oxygen, imino, iminium, lower alkyliminium, or —NHR^(c) where R^(c) is as defined above, and in generalformula VIII, R⁶ is hydrogen or —NHR^(c) where R^(c) is as definedabove. In particular embodiments, X¹ is oxygen, R³, R⁴, R⁵, R⁸ (ifpresent), R⁹, R¹², R¹⁵, R¹⁷, R¹⁸, R¹⁹, R²⁰, and R²² are hydrogen; R⁷ (ifpresent) is hydrogen or halogen; R¹⁰ and R¹¹ independently are hydrogen,amino, lower alkyl, carboxyl, or —SO₃H; R¹³ is hydrogen, lower alkyl,lower alkoxy, —SO₃H, or —COOR¹⁴ where R¹⁴ is hydrogen or lower alkyl;R¹⁶ is hydroxyl, amino, lower alkyl iminium, or —NHR^(c) where R^(c) isas defined above; R²¹ is oxygen, imino, iminium, or lower alkyl iminium;R⁶ is oxygen, imino, iminium, lower alkyl iminium, or —NHR^(c) whereR^(c) is as defined above in general formula VII, or R⁶ is hydrogen or—NHR^(c) where R^(c) is as defined above in general formula VIII. Incertain embodiments, if X¹ is oxygen and either R¹⁶ or R¹⁸ is hydroxylin structure VII, then R⁶ is other than oxygen, amino, or alkyl amino,or at least one of R³, R⁸ or R¹⁵ is other than hydrogen, or R¹³ is oneor more atoms forming a ring system with rings B and D and the bonddepicted as “

” in ring B is a single bond. Representative compounds according togeneral formulas VII and VIII are shown in Table 3.

TABLE 3 Cpd R⁶* R⁷ R¹⁰ R¹¹ R¹³ R¹⁶ R¹⁸ R¹⁹ R²¹  1a O H H H —OCH₃ —OH H ——  1b O H —CH₃ H —CH₃ —OH H — —  1c O H H —COOH H —OH H — —  1d O H H—NH₂ H —OH H — —  1e O H H H —SO₃H —OH H — —  1f O H —SO₃H H —SO₃H —OH H— —  1g O F H H H —OH H — —  1h ═NH H —SO₃H H —SO₃H —NH₂ H — —  1i═N(CH₃)₂ ⁺ H H H H —OH H — —  1j ═N(CH₃)₂ ⁺ H H H H —N(CH₃)₂ H — —  1k═N(CH₃)₂ ⁺ H —SO₃H H —SO₃H —N(CH₃)₂ H — —  6a H — —CH₃ H —CH₃ —OH H H ═O 6b H — —SO₃H H —SO₃H —OH H H ═O  6c H — —CH₃ H —CH₃ —NH₂ H H ═NH  6d H— —CH₃ H —CH₃ —N(CH₃)₂ H H ═N(CH₃)₂ ⁺  6e H — H —COOH H —OH H H ═O  6f H— H —COOH H —N(CH₃)₂ H H ═N(CH₃)₂ ⁺ 36a H — H —COOH —CH₃ H —OH ═O H 36bH — H H —COOH H —OH ═O H 36c H — H —COOH —CH₃ H —OH ═N(CH₃)₂ ⁺ H 36d H —H H —COOH H —OH ═N(CH₃)₂ ⁺ H *All R substituents not included in Table 3are hydrogen and X¹ is oxygen.

In some embodiments, the NIR dyes have mixed annulation, e.g., type [a]and type [c], type [a] and type [b], or type [b] and type [c]. Forexample, in some embodiments, R³ and R⁴ together form an aryl orsubstituted aryl ring and R⁷ and R⁸ together form an aryl or substitutedaryl ring as shown in general formula IX. In other embodiments, R³ andR⁴ together form an aryl or substituted aryl ring and R⁶ and R⁷ togetherform an aryl or substituted aryl ring as shown in general formula X. Instill other embodiments, R¹ and R² together form an aryl or substitutedaryl ring and R⁶ and R⁷ together form an aryl or substituted aryl ringas shown in general formula XI.

In general formulas IX-XI, each bond depicted as “

” is a single or double bond as needed to satisfy valence requirements.R¹-R⁵, R⁷-R²² and X¹ are defined as above, and R⁶ is hydrogen, hydroxyl,oxygen, halogen, thiol, amino, alkyl amino, or —NHR^(c) where R^(c) isas defined above. In certain embodiments, X¹ is oxygen; R¹-R⁶ (ifpresent), R⁸ (if present), R⁹, R¹⁰, R¹², R¹⁵, R¹⁷, and R²² are hydrogen;R¹¹ is hydrogen, amino, lower alkyl, carboxyl, or —SO₃H; R¹³ ishydrogen, lower alkyl, lower alkoxy, —SO₃H, —COOR¹⁴ where R¹⁴ ishydrogen or lower alkyl and the bond depicted as “

” in ring B is a double bond, or R¹³ is one or more atoms forming a ringsystem with rings B and D and the bond depicted as “

” in ring B is a single bond; R¹⁶, R¹⁷, and R¹⁸ independently arehydrogen, hydroxyl, oxygen, lower alkoxy, amino, alkyl amino, or—NHR^(c) where R^(c) is as defined above, and at least one of R¹⁶ andR¹⁸ is other than hydrogen; R¹⁹-R²¹ independently are hydrogen,hydroxyl, thiol, oxygen, imino, iminium, alkyl imino, alkyl iminium,amino, alkyl amino, or —NHR^(c) where R^(c) is as defined above, and atleast one of R¹⁹-R²¹ is other than hydrogen. In particular embodiments,X¹ is oxygen; R¹-R⁶ (if present), R⁸ (if present) R⁹, R¹⁰, R¹², R¹⁵,R¹⁷, and R²² are hydrogen; R¹¹ is hydrogen or carboxyl; R¹³ is loweralkyl or carboxyl; R¹⁶ and R¹⁸ independently are hydrogen, hydroxyl, oroxygen, or —NHR^(c) where R^(c) is as defined above, and at least one ofR¹⁶ and R¹⁸ is other than hydrogen; R¹⁹-R²¹ independently are hydrogen,oxygen alkyl iminium, or —NHR^(c) where R^(c) is as defined above, andat least one of R¹⁹-R²¹ is other than hydrogen. Representative compoundsaccording to general formulas IX-VI are shown in Table 4.

TABLE 4 Cpd R¹¹* R¹³ R¹⁶ R¹⁸ R¹⁹ R²⁰ R²¹ 33a —COOH —CH₃ H —OH H H ═O 33bH —COOH H —OH H H ═O 33c —COOH —CH₃ H —OH H H ═N(CH₃)₂ ⁺ 33d H —COOH H—OH H H ═N(CH₃)₂ ⁺ 35a —COOH —CH₃ H —OH ═O H H 35b H —COOH H —OH ═O H H35c —COOH —CH₃ H —OH ═N(CH₃)₂ ⁺ H H 35d H —COOH H —OH ═N(CH₃)₂ ⁺ H H 37a—COOH —CH₃ H —OH H ═O H 37b H —COOH H —OH H ═O H 37c —COOH —CH₃ H —OH H═N(CH₃)₂ ⁺ H 37d H —COOH H —OH H ═N(CH₃)₂ ⁺ H 38a —COOH —CH₃ —OH H H ═OH 38b H —COOH —OH H H ═O H 38c —COOH —CH₃ —OH H H ═N(CH₃)₂ ⁺ H 38d H—COOH —OH H H ═N(CH₃)₂ ⁺ H 39a —COOH —CH₃ —OH H ═O H H 39b H —COOH —OH H═O H H 39c —COOH —CH₃ —OH H ═N(CH₃)₂ ⁺ H H 39d H —COOH —OH H ═N(CH₃)₂ ⁺H H *All R substituents not included in Table 4 are hydrogen and X¹ isoxygen.

B. Structural Effects on Spectra and Stokes Shifts

Systematic structure-based evaluation surprisingly demonstrated thatshifting the position of a substituent on the annulated ring of a singlyannulated xanthene substantially red-shifted the emission and absorptionmaxima. For example, in some embodiments as shown in FIG. 1, type [c]annulated xanthenes according to general formula III may includesubstituents at the 3-position of the annulated ring (R¹⁶) or at the1-position (R¹⁸). In working embodiments, positioning the substituent atthe 1-position (R¹⁸) instead of the 3-position (R¹⁶) in general formulaIII and IV was found to red-shift the absorbance spectrum by more than10 nm, typically by more than 30 nm or more than 50 nm, such as by10-200 nm, 10-60 nm, 30-75 nm, 50-75 nm, 30-100 nm, or 75-200 nm. Insome embodiments, the 3-1 transposition red-shifted the emissionspectrum by at least 100 nm, such as by 100-200 nm, 120-160 nm, or130-150 nm. In certain embodiments, when the substituent was at the1-position (R¹⁸ in general formulas III and IV), the emission maximumoccurred at greater than 725 nm, greater than 750 nm, or greater than770 nm. In some instances, the emission maxima were not measurable, butare believed to occur at greater than 850 nm (beyond the range of theinstrument). Simultaneous enhancement of the Stokes shift also wasnoted. In some embodiments, the 3-1 transposition increased themagnitude of the Stokes shift by greater than 50 nm, such as by 50-100nm, 70-90 nm, or 70-100 nm.

Thus, the 3-1 transposition provides several advantages. Both absorbanceand emission maxima are red-shifted. Because the emission spectratypically have a greater red shift than the absorbance spectra, theStokes shift is enhanced. This behavior occurs across symmetric (e.g.,general formula IV) and asymmetric structures (e.g., general formulaIII), and was observed across various ionizable groups (e.g., hydroxyland amine functionalities). In some embodiments, combining the 3-1transposition with transposition of ionizable functionalities alsoprovides an unexpected pH response. Some rhodols absorb and emit longwavelengths at high pH. However, with some embodiments of the disclosedfluorophores that combine a 3-1 transposition with ionizable moietytransposition, long wavelength behavior was observed at acidic pHvalues.

In some embodiments, anionic forms of the fluorophores having a 3-1transposition produce a greater red shift than the corresponding neutralforms of the fluorophores. For example, the absorbance maximum ofcompound 15a is red-shifted 15 nm compared to compound 20a. However, theabsorbance maximum of compound 15h, the anionic form of compound 15a,has a red-shift of 50 nm compared to compound 20c, the anionic form ofcompound 20a. FIG. 24 is a series of UV-visible spectra illustrating theeffect of ionization on the absorption spectra of some embodiments ofthe disclosed fluorophores. The bathochromic shift in emissionwavelengths for compound 15h compared to compound 20c was 130 nm. Thedifferences between these shifts results in NIR emission at 760 nm witha significantly enhanced Stokes shift of 160 nm.

There are through-space polar interactions between R¹⁸ and the internalbridge oxygen in compounds according to general formulas III, IX, and X.The field effect is further enhanced in the case of symmetric compounds,such as those according to general formula IV when R¹⁹ is oxygen. Aunique intramolecular hydrogen-bonding network also is present in suchcompounds, e.g., compound 8 (FIG. 25). This network produces enhancednegative charge density on the ionizable oxygen atoms, thereby promotinga relatively large bathochromic shift. Molecular simulations demonstratethat the hydrogen bonding network also enhances coplanarity of the fusedring system, which is diminished when the hydroxyl group is deprotonated(FIG. 26). The hydrogen bonding can be seen in compounds such ascompound 8 by ¹H NMR spectroscopy. A redistribution of the electronicdensity around the hydrogen atom occurs upon hydrogen bond formationbetween the hydroxyl proton, the carbonyl, and the central oxygen ofcompound 8. In compound 8, simulations show that the total hydrogen bonddistance O—H—O between the carbonyl at R¹⁹ and the hydroxyl at R¹⁸ is2.52 Å, which is lower than the typical hydrogen bonding distance (≥2.8Å) and close to the distance observed for low-barrier hydrogen bonds(2.55 Å). Evidence of hydrogen bonding was also seen in compound 15a,but to a lesser degree. In compound 15a, the total hydrogen bonddistance O—H—O between the hydroxyl at the R¹⁸ position and the xanthenecentral oxygen is 2.95 Å, which falls in range of common hydrogen bonds.Removal of the shared proton in compound 8 to produce compound 8a doesnot result in a large bathochromic shift in the absorption maximum as isobserved when compound 22 is deprotonated to compound 22a, thusproviding further evidence of the effect of the hydrogen-bonding networkon the characteristics of compounds such as 8. Surprisingly, whencompound 10 was deprotonated, the shift was hypsochromic.

In some embodiments, e.g., compound 8, NIR fluorescence was observed incompounds exhibiting hydrogen bonding up to pH 9, while removal of theshared proton was observed to quench the fluorescence. Notably, compound7 exists as its corresponding colorless lactone in 1:9 DMSO:buffer belowphysiological pH of 7.4, and thus embodies a unique pH probe.

Some embodiments of fluorophores having a chemical structure accordingto general formula III or IV have longer absorption and emissionwavelengths than rhodamine. Analogues having an —NHR^(c) (boronic acid)substituent at R¹⁶ and R⁶ (formula III) or R²¹ (formula IV) are furtherred-shifted that the corresponding fluorophores having an —NH₂ at R¹⁶and R⁶/R²¹. In the absence of sugars, the boronic acids may be quenchedthrough a PET (photoinduced electron transfer) mechanism. In certainembodiments, bathochromic shifts and increased quantum yields wereobserved for both asymmetric (formula III) and symmetric (formula IV)bis-boronic acid analogues upon sugar binding.

IV. SYNTHESIS

Some embodiments of naphthofluorescein analogues having a chemicalstructure according to general formula IV are synthesized as shown inScheme 1 (FIG. 2). In some embodiments, analogues are synthesized byacid condensation of 1,8-dihydroxynaphthalene and phthalic acid inmethanesulfonic acid to produce a lactone-containing compound (compound7). Reacting compound 7 with acetyl chloride in methanol opens thelactone ring, thereby esterifying the compound and producing the methylcarboxylate 8; one of the hydroxyl groups is converted to an oxygenthrough electronic density redistribution in the conjugated system.Further reaction of compound 8 with potassium carbonate and methyliodide in dimethyl formamide converts the remaining hydroxyl group to amethoxy group (compound 9). In other embodiments, analogues according togeneral formula IV are synthesized by acid condensation of1,8-dihydroxynaphthalene with a desired aldehyde in phosphoric acid toproduce analogues having structures according to representativecompounds 10 and 11. In some embodiments, compounds 10 and 11 furtherare reacted with potassium carbonate and methyl iodide to produce methylanalogues.

Analogues having a chemical structure according to general formula IIIare synthesized by acid condensation as shown in Scheme 2 (FIG. 3). Ahydroxybenzophenone derivative (compound 12) and a 1,8-naphthalenederivative (compound 13) are condensed by reaction in a 1:1 mixture ofmethanesulfonic acid and trifluoroacetic acid to produce alactone-containing compound (compound 14). Further reaction of compound14 with methanol in sulfuric acid opens the lactone ring, therebyesterifying the compound and producing the methyl ester (compound 15);compound 14 is converted to compound 15 via electronic redistribution inthe conjugated system. Alternatively, reacting compound 14 withpotassium carbonate and methyl iodide opens the ring and producescompound 16.

Certain asymmetric seminaphthofluorescein and rhodamine analogues havinga chemical structure according to general formula III are synthesized asshown in Scheme 3 (FIG. 4). A hydroxybenzophenone derivative (compound17) and a naphthol derivative (compound 18) are reacted via acidcondensation in methanesulfonic acid/trifluoroacetic acid to produce alactone-containing compound, compound 19. Further reaction of compound19 with methanol in sulfuric acid produces compound 20a.

A commercially available, lactone-containing naphthofluorescein analoguewas converted to its methyl ester via Fischer esterification as shown inScheme 4 (FIG. 5). The naphthofluorescein analogue (compound 21) isdissolved in methanol. Sulfuric acid is added, and the mixture isrefluxed for 24 hours to produce compound 22.

Some analogues having chemical structures according to general formulaVI and some asymmetric, extended-conjugation analogues having chemicalstructures according to general formulas VIII-XI are synthesized asshown in Scheme 5 (FIG. 6). The method shown in Scheme 5 involves theformation of tertiary carbinol leuco bases via a Grignard reaction,followed by deprotection and condensation with BBr₃ to produce thecorresponding xanthene dyes. Ketone precursors can be synthesized byreaction of the corresponding methoxy-naphthalenes with benzoylchlorides or phthalic anhydride in the presence of polyphosphoric acid(Gorelick et al., Zhurnal Org. Kihimii, 1983, 19, 199-206).

Some analogues having chemical structures according to general formula Vand VII are synthesized as shown in Scheme 6 (FIG. 7). Ahydroxybenzophenone derivative is synthesized and condensed with a1,8-naphthalene derivative in in a 1:1 mixture of methanesulfonic acidand trifluoroacetic acid to produce a mixture of lactone-containingcompounds. The lactone rings can be opened, if desired, by reacting thecompounds with methanol in sulfuric acid, or by reacting the compoundswith potassium carbonate and methyl iodide to produce methyl esterderivatives.

Some analogues having chemical structures according to general formula Iwhere X¹ is Si(CH₃)₂, Ge(CH₃)_(2,) or Sn(CH₃)₂ may be synthesizedaccording to Scheme 7 (FIG. 8). With respect to Scheme 7, R¹, R², R⁴,R⁵, R⁷, and R⁸ are as previously defined, with the proviso that none ofR¹, R², R⁴, R⁵, R⁷, or R⁸ is —NH_(2.) In step (a), a dilithiumintermediate may be generated from a bis(2-bromophenyl)methanederivative by halogen-metal exchange reaction, quenched with theappropriate dialkyl dichloride of silicon, germanium, or tin, andoxidized to form a xanthene. In step (b), a phenyl lithium derivativemay be inserted to produce a rhodol or rhodamine-type structure. See,e.g., Koide et al., ACS Chem. Biol. 2011, 6(6), 600-608.

Some analogues having chemical structures according to general formula Iwhere X¹ is S or Se may be synthesized according to Scheme 8 (FIG. 9).With respect to Scheme 8, R¹, R², R⁴, R⁵, R⁷, and R⁸ are as previouslydefined, with the proviso that none of R¹, R², R⁴, R⁵, R⁷, or R⁸ is —NH₂or halo. A sulfur analogue may be synthesized by preparing 3-iodoanisolefrom m-anisidine via its diazonium chloride, and separately preparing3-methoxythiophenol sodium salt by reaction of sodium ethoxide with acorresponding thiophenol. Heating 3-iodoanisole and 3-methoxythiophenolsodium salt with powdery copper produces 1,1′-thiobis(3-methoxybenzene,which is converted to m,m′-thiodiphenol by ether cleavage with hydrogeniodide in acetic acid. A rhodol analogue may then be prepared by meltingm,m′-thiodiphenol with phthalic anhydride in the presence of zincchloride (path a), or by melting with 2-sulfobenzoic acid cyclicanhydride and toluene-4-sulfonic acid (path b) to produce thesulfobenzoic acid analogue shown in FIG. 9. See, e.g., Pfoertner, J.Chem. Soc., Perkin Trans. 2, 1991, 523-526.

Some analogues having chemical structures according to general formula(III) where R⁶ and R¹⁶ are amino or boronic acid are synthesizedaccording to Scheme 9 (FIG. 10). A rhodamine is hydrolyzed under basicconditions, followed by acid condensation with anaminohydroxynaphthalene to produce a seminaphthorhodamine. Reductiveamination produces the corresponding bis-boronic acid.

Some analogues having chemical structures according to general formula(iv) where R¹⁶ and R²¹ are amino or boronic acid are synthesizedaccording to Scheme 10 (FIG. 11). A naphthorhodamine is prepared byacid-promoted condensation of phthalic anhydride and anaminohydroxynaphthalene. Reductive amination produces the correspondingbis-boronic acid.

In some embodiments, it may be advantageous to reduce the dye pKa and/orto increase aqueous solubility of the dyes. In certain embodiments, pKamay be reduced by halogenating the dye, such as by position one or morefluorine atoms ortho to one or more ionizable moieties such that amajority of the dye molecules are in an ionic form in a neutral aqueoussolution. The inventors have discovered that ionized species of thedisclosed dyes generally have a larger Stokes shift than neutral dyemolecules. Embodiments of the dyes may be fluorinated by using afluorinated naphthol during the synthesis, i.e., during the condensationreaction. Fluorinated naphthols may be synthesized by reactinghydroxynaphthalenes with a fluorine donor (e.g., Selectfluor®(1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octanebis(tetrafluoroborate)) in methyl cyanide at reflux as described in theliterature (Yang et al., Heteroatom Chem., 1998, 9, 229-239; Bluck etal., J. Fluor. Chem., 2004, 125, 1873-1877).

In some embodiments, it also may be advantageous to reduce non-specificbinding in biological media. To reduce non-specific binding,oligopegylated derivatives of the disclosed dyes can be prepared byconjugating the dye to an oligoethylene glycol. If bi-functionalpegylating reagents are used, the dye may further be conjugated to otherbiological molecules of interest. Suitable bi-functional pegylatingreagents comprising 6-8 ethylene glycol units are commercially availableor can be prepared using literature protocols (Svedhem et al., J. Org.Chem., 2001, 66, 4494-4503; Wosnick et al., J. Am. Chem. Soc., 2005,127, 3400-3405). Pegylated reagents can be conjugated via amide bondformation to embodiments of the disclosed dyes that comprise acarboxylic group. Pegylation also may increase solubility of dyes havinghydrophobic properties.

V. APPLICATIONS

Some embodiments of disclosed NIR dyes are suitable for use inchallenging biological media such as blood, plasma, and urine. Blood hasstrong background absorption, significant autofluorescence, and scatterin the visible region. Certain embodiments of the disclosed NIR dyeshave emission spectra maxima at wavelengths that are long enough toovercome interference from blood hemoglobin, e.g., an emission spectrummaximum at greater than 640 nm, or greater than 680 nm. In particularembodiments, the disclosed NIR dyes also have an absorbance spectrummaximum that is greater than 640 nm, such as greater than 680 nm. Insuch embodiments, a light source having a wavelength greater than 640 nmcan be utilized, thereby minimizing light absorption by blood. Dyes thatfunction beyond the optical range of blood will simplify testing foranalytes and biomarkers by limiting dilution and samplepreparation/handling, thereby reducing sources of error, reducing timeto obtain results, and/or reducing health hazards due to sample handlingand manipulation. Current methods and detection agents utilizechromatography, fragile materials (e.g., those that are unstable inaqueous solution, require storage below −20° C. and/or require storagein the dark), and/or a high degree of sample processing. For example, incurrent methods, samples may be diluted more than 1000-fold to overcomeoptical interference from blood, or testing may be performed in plasmainstead of whole blood. In contrast, some embodiments of the disclosedNIR dyes are stable for weeks at ambient temperature, even in solution.Certain embodiments of the disclosed NIR dyes also are more photostablein cell culture media than fluorescein.

In particular, some embodiments of the disclosed NIR dyes includefunctional groups that may facilitate detection of specific molecularbiomarkers. Embodiments of the NIR dyes may be functionalized to (a)produce a desired geometry having a combination of covalent and/orsupramolecular interactions between the dye and a desired biomarkerand/or (b) to alter oxidation-reduction and/or energy transferproperties in the presence of a desired biomarker. For instance,molecular modeling also indicates that some embodiments having achemical structure according to general formula VI and polar end groupssuch as amino, oxygen, and/or hydroxyl groups at positions R¹⁷ and R²⁰may exhibit favorable electrostatic interactions with the polar ends ofglutathione (FIG. 12).

Molecular modeling also indicates that some embodiments of the NIRfluorophores having a chemical structure according to general formula VIwherein at least one of R¹⁷ and R²⁰ is —NHR^(c) where R^(c) is

may preferentially interact with specific nucleosides (FIG. 13). Incertain embodiments, specificity may be enhanced when R¹³ comprises acarboxylate moiety.

Biomarkers of interest include glutathione (GSH), which is diminished inwhole blood, leukocytes and plasma in mitochondrial disorders (e.g.,diabetes, atherosclerosis, neurodegenerative diseases, hypoxic-ischemicencephalopathy, autism, retinopathy of prematurity, chronic progressiveexternal ophthalmoplegia, and cancer (Akturi et al., PNAS U.S.A., 2009,106, 3941-3945)) and organic acidemias (e.g., methylmalonic acidemia).Some commercially available methods for detecting GSH utilize detectionagents that are not selective for GSH over other thiols (e.g., cysteine,dithiothreitol) and/or require testing in plasma. However, GSH levels inwhole blood are in the millimolar range, or 100-1000 fold higher than inplasma.

Embodiments of the disclosed NIR dyes may be suitable for selectivedetection of GSH in blood. For example, molecular modeling shows thatcompound 5a (Table 2) has very favorable structural interactions withGSH. Additionally, compound 5a was found to be detectable in a 5% bloodsolution (see Example 2). Embodiments of NIR dyes functionalized topromote multipoint, glutathione-selective covalent, supramolecularand/or redox interactions may serve as indicators for oxidative stressand mitochondrial disorders.

Rhodamine B lactol has GSH selectivity, which is thought to arise fromreaction with the aldehyde tautomer, thereby affording the observedemission and absorbance increases.

It is believed that the equilibrium favors the thiohemiacetal in thecase of GSH due, at least in part, to favorable electrostaticinteractions between the polar groups of the dye (which actuallymodulate the ionization state and optical properties) and those of theGSH tripeptide. In order to understand potential salt bridge formationand ion pairing, the covalent complexes formed in the reaction of dyessuch as compound 24 were simulated, demonstrating that the relativelyextended GSH moiety can interact with the polar ends of the dyessimultaneously (FIG. 12).

Other biomarkers of interest includesuccinyl-5-amino-4-imidazole-carboxamide riboside (SAICA-riboside) andsuccinyladenosine (S-Ado), which are indicators of adenylosuccinatelyase (ADSL) deficiency.

ADSL deficiency is a rare (approximately 1 in 200,000) inborn error ofpurine metabolism, which can result in mental retardation and seizure(Jaeken, Lancet, 1984, 2, 1058-1061). Undiagnosed genetic defects inpurine and pyrimidine (PP) metabolism may result in early death and/orinstitutionalization. ADSL deficiency is characterized by massiveurinary excretion (millimolar levels) of SAICA-riboside and S-Ado, thenucleosides corresponding to SAICA-ribotide (SAICAr) andadenylosuccinate.

Embodiments of the disclosed dyes functionalized with covalent andsupramolecular binding sites targeted for specific nucleosides may serveas indicators for ADLS deficiency. In some embodiments, the NIR dyeincludes at least one boronic acid functional group, i.e., at least oneR group is —NHR^(c) where R^(c) is

A rhodamine modified with a phenyl boronic acid can exhibitunprecedented affinity for ribose and congeners as compared to fructose(Jiang et al., J. Am. Chem. Soc., 2006, 128, 12221-12228).

Computer-assisted molecular simulations indicate that, apart from thepreference of the boronic acid to react with the 2,3-cis diol of thefuranose form of ribose and the strong electrostatic interactionB—O—H—N⁺, non-covalent secondary interactions play an important rolemodifying the ionization state of the chromophore. FIG. 13 illustratesthe charged H-bonding that can occur between the nucleosides and thecarboxylate moiety of two embodiments (compounds 43 and 44, Table 2) ofdyes having chemical structures according to general formula VI.Energy-minimized models in FIG. 13 were prepared using simulatedannealing (sequential cycles of heating/cooling) using Sybyl™ X 1.1(Tripos, St. Louis, Mo.) and geometry optimization with MOPAC2009(Stewart Computational Chemistry, Colorado Springs, Colo.). Interactionwith the carboxylate moiety appears to play a significant role in theinteractions with S-Ado and SAICAr since esterification of thecarboxylate moiety in the rhodamine bis-boronic acid causes itsselectivity to revert to fructose.

In some instances, if evaluation in biological media is unsatisfactory,a molecule-imprinted polymer (MIP) specific to a particular analyte maybe synthesized and utilized in a solid-phase extraction step prior toevaluation with embodiments of the disclosed NIR dyes. The MIP willallow capture and concentration of the analyte. Excellent synergism maybe achieved as any deficiency in selectivity by the fluorophore-viologenconjugates and/or the MIP may compensate for the other component.

Some embodiments of the disclosed NIR dyes may be useful for cellimaging, intracellular pH sensing (e.g., by evaluating the absorbanceand/or emission spectra and identifying dye ionization states within thecell), and/or detection of intracellular analytes such as thiols,hydrogen peroxide, hydrogen sulfide, and/or cyanide.

VI. KITS

Kits are also a feature of this disclosure. Embodiments of the kitsinclude at least one compound according to any one of general formulasI-XI and suitable for selectively detecting an analyte in a sample(e.g., a biological fluid such as blood or urine). In some embodiments,the kits also include at least one buffer solution in which thecompound, when combined with a sample including, or suspected ofincluding, an analyte, will undergo a change in its absorbance spectrumand/or emission spectrum compared to the compound in the buffer solutioncombined with a sample that does not include the analyte. The kits mayinclude a color comparison chart for evaluating a color change producedby a reaction between the compound and the analyte. The kits also mayinclude one or more containers, such as a disposable test tube orcuvette, in which the detection can be performed. The kits may furtherinclude instructions for performing the detection. In some embodiments,the kits include control samples of analytes, e.g., glutathione,cysteine, homocysteine, succinyl-5-amino-4-imidazolecarboxamideriboside, and/or succinyladenosine. Typically the control samples areprovided in solid form.

In some embodiments of the kits, the compound is provided as a solid,and the buffer is provided in liquid form. The buffer may be provided ata concentration suitable for detecting Cys, Hcy, GSH,succinyl-5-amino-4-imidazolecarboxamide riboside, succinyladenosine or amixture thereof. Alternatively, the buffer may be provided as aconcentrated solution, which is subsequently diluted prior to use. Incertain embodiments, the compound may be premeasured into one or morecontainers (e.g., test tubes or cuvettes), and the detection issubsequently performed by adding the buffer and test sample to thecontainer.

VII. EXAMPLES

Reagents and General Procedures:

-   Chemistry. Unless otherwise indicated, all commercially available    starting materials were used directly without further purification.    Naphthofluorescein was obtained from Sigma-Aldrich. Silica gel    (Sorbent Technologies) 32-63 μm was used for flash column    chromatography. ¹H-NMR was obtained on an ARX-400 Advance Bruker    spectrometer. Chemical shifts (δ) are given in ppm relative to    d₆-DMSO (2.50 ppm, ¹H, 39.52 ¹³C) unless otherwise indicated. MS    (HRMS, ESI) spectra were obtained at the Portland State University    Bioanalytical Mass Spectrometry Facility on a ThermoElectron    LTQ-Orbitrap high resolution mass spectrometer with a dedicated    Accela HPLC system.-   General acid condensation using methanesulfonic acid.    Dihydroxynaphtahlene (3.12 mmol) and phthalic anhydride (1.56 mmol)    are dissolved in 3 mL of methanesulfonic acid. The mixture is    stirred at 90° C. for 24 hours. The mixture is allowed to cool down    to room temperature, then poured into distilled water (50 mL). The    precipitate is filtered and washed with water (3×50 mL). If no    precipitate is formed, the mixture is neutralized to pH 5-7 by    portion-wise addition of solid NaHCO_(3.) The precipitate is dried    under vacuum. The target compound is isolated by flash column    chromatography on silica gel.-   General condensation method using CH₃SO₃H:TFA 1:1 mixture.    Hydroxybenzophenone (918 μmol) and 1,8-naphthalene derivative (1380    μmol) are dissolved in 1.5 mL of methanesulfonic acid, then 1.5 mL    of trifluoroacetic acid (TFA) are added. The mixture is heated and    stirred at 80° C. for 16-24 hours. The reaction mixture is allowed    to warm to room temperature, and then poured into 50 mL of    deionized (DI) water. The mixture is neutralized to pH 6-7 by    portion-wise addition of solid NaHCO_(3.) The resulting precipitate    is filtered, washed with DI water and air dried. The target compound    is isolated by flash column chromatography on silica gel.-   General esterification method A. Carboxylate (0.243 μmol) is    dissolved in 2 mL of methanol (MeOH). To this solution is added    concentrated H₂SO₄ (100 μL) dropwise, then the mixture is refluxed    24 hours. The mixture is allowed to cool down to room temperature,    then poured into 50 mL of ice water and 200 mg of NaHCO₃ is added in    one portion. If a precipitate forms, the solid is filtered and    washed with 2% NaHCO₃ (2×10 mL), then with water (2×10 mL). If no    precipitate is obtained, the neutralized aqueous phase is extracted    with CHCl₃ (3×50 mL). The organic phase is dried over Na₂SO₄ and the    solvent evaporated under vacuum. The target compound is then    isolated by flash column chromatography.-   General esterification method B. Under an argon atmosphere, the    compound (0.131 mmol) is dissolved in 25 mL of anhydrous methanol.    The solution is cooled to 0° C. in an ice bath. Acetyl chloride (750    μL) is added dropwise. The mixture is stirred and kept at 50° C. for    48 hours. Acetyl chloride (300 μL) is added dropwise, and the    mixture kept at 50° C. for additional 24 hours. The mixture is    allowed to cool down to room temperature, and the solvent is    evaporated under vacuum.

Example 1 Syntheses and Characterization

As shown in Scheme 1, analogues having a structure according to generalformula IV were synthesized either via acid condensation of1,8-dihydroxy-naphthalene and phthalic anhydride in methanesulfonic acidor by condensation of 1,8-dihydroxynaphthalene with the correspondingaldehydes in 85% H₃PO₄ at 125° C. /24 hours. The naphthofluoresceinmethyl ester 22 was obtained via a typical Fischer esterificationprotocol from 21 (Scheme 4). Asymmetric seminaphthofluorescein, rhodoland rhodamine analogues according to general formula III (Schemes 2 and3) were synthesized by acid condensation of hydroxybenzophenones withthe corresponding naphthols in a mixture of CH₃SO₃H:TFA 1:1 at 80° C.for 16-24 hours. The hydroxybenzophenones and 1,8-naphthalenederivatives required were synthesized according to described or modifiedliterature protocols. The methyl ester derivatives were obtained byesterification in MeOH catalyzed by either H₂SO₄ or HCl; further methylalkylation was furnished by treatment of either the carboxylate ormethyl ester intermediate with methyl iodide in the presence of K₂CO₃ indimethylformamide. In general, overall good yields were obtained formost of the compounds included in this series with the exception of thecondensation products between dihydroxybenzophenone and 8-amino naphtholderivatives, where the major isolated product corresponds tofluorescein. All compounds were isolated by flash column chromatography(normal or reversed phase) and characterized by NMR and MS. Thestructure of compound 14a (FIG. 14) was confirmed by X-ray singlecrystal structure.

Compounds 21 and 7 (type [c] fully annulated naphthofluorescein withknown regiochemistry versus fully annulated type [c] naphthofluoresceinwith 3-1 transposition)

Compounds 21 and 7 were characterized by UV-visible spectroscopy (FIG.15). Absorbance was measured using 15 μM solutions of each compound in asolution that was 90% aqueous, 10% DMSO. The peaks at 600 nm (lightdashed line) and 675 nm (light solid line) represent the dianions ofcompounds 21 and 7, respectively, in NaOH (pH 12). The peaks at 590 nm(heavy dashed line) and 650 nm (heavy solid line) represent a mix ofmono- and dianions of compound 21 at pH 7.4 and predominantly monoanionsof compound 7 at pH 9, respectively. Thus, the 3-1 transpositionresulted in red-shifted absorption spectra of about 60-75 nm, dependingon the ionization state.

Compounds 21 and 7 also were characterized by their fluorescenceemission spectra. Fluorescence was measured using 15 μM solutions ofeach compound in a solution that was 90% aqueous, 10% DMSO. For compound21, the longest wavelength emission observed from the dianions occurrednear 680 nm at pH 9. For compound 7 (transposed), the longest wavelengthemission observed from the monoanion occurred near 800 nm at pH 9.Emission from the transposed dianion in sodium hydroxide was either tooweak to be detected or, more likely, beyond the working range of theinstrument (>850 nm). The 3-1 transposition resulted in red-shiftedemission of about 120 nm, and possibly more depending on the ionizationstate.

The Stokes shift of each compound was measured and determined to beabout 80 nm for the compound 21 dianion, and about 150 nm for thecompound 7 (transposed) monoanion. Thus, the 3-1 transposition alsoenhanced the Stokes shift.

Compounds 22 and 8 (type [c] fully annulated naphthofluorescein methylester with known regiochemistry versus fully annulated type [c]naphthofluorescein methyl ester with 3-1 transposition)

Compound 22 was synthesized according to Scheme 4. Naphthofluorescein(compound 21, 0.05 g, 0.115 mmol) was dissolved in 2 mL methanol. Thesolution was cooled to 0° C. in an ice bath. Concentrated sulfuric acid(100 μL) was added to the solution, and the mixture was refluxed for 24hours. The mixture was allowed to cool to room temperature, and thenpoured into 250 mL of ice water; 200 mg NaHCO₃ was added. Theprecipitate was filtered and washed twice with 2% NaHCO₃ and then twicewith water. Compound 22 was isolated by flash column chromatography onsilica gel using CH₂Cl₂:MeOH 9:1 for elution. The yield was 26 mg, 50%.¹H NMR (400 MHz, DMSO) δ 10.05 (s, 1H), 8.96 (d, J-9.2 Hz, 2H), 8.60 (d,J-9.0 Hz, 1H), 8.33 (d, J=7.9 Hz, 1H), 7.98 (t, J=6.9 Hz, 1H), 7.89 (t,J=7.7 Hz, 1H), 7.63 (d, J=9.2 Hz, 2H), 7.56 (d, J=6.8 Hz, 1H), 7.32-7.21(m, 1H), 7.18-7.03 (m, 2H), 6.91 (d, J=9.1 Hz, 2H), 3.56 (s, 3H). HR ESI[M+H⁺] m/z 447.1231 calc for C₂₉H₁₉O₅; 447.1237.

Compound 8 was synthesized by dissolving 1,8-dihydroxy-naphthalene (0.5g, 3.12 mmol) and phthalic anhydride (0.231 g, 1.56 mmol) in 2 mL ofmethanesulfonic acid, and then adding 2 mL trifluoroacetic acid. Themixture was heated and stirred at 80° C. for 24 h. The mixture wasallowed to cool to room temperature, and then poured into 100 mL ofdeionized water. The precipitate (compound 7) was filtered and washedwith deionized water, and then isolated by flash column chromatographyon silica gel using CH₂Cl₂:MeOH 9:1 for elution. Yield 0.122 g, 18%.Esterification then was carried out as described for compound 22. Thetarget compound was isolated by flash column chromatography on silicagel using CH₂Cl₂:MeOH 9:1 for elution. Yield 0.023 g, 86%. ¹H NMR (400MHz, DMSO) δ 14.45 (s, 1H), 8.32 (dd, J=7.9, 1.0 Hz, 1H), 7.97 (td,J=7.5, 1.3 Hz, 1H), 7.87 (td, J=7.7, 1.3 Hz, 1H), 7.62-7.66 (m, 4H),7.56 (dd, J=7.6, 0.9 Hz, 1H), 7.13, (d, J=7.2 Hz, 2H), 6.91 (dd, J=8.4,0.8 Hz, 2H), 6.77 (d, J=9.1 Hz, 2H), 3.55 (s, 3H). ¹³C NMR (101 MHz,DMSO) δ 167.48, 165.22, 154.58, 137.69, 135.10, 134.52, 133.66, 130.95,130.56, 130.40, 130.05, 129.20, 120.79, 119.99, 119.87, 116.26, 112.80,52.42. HR ESI [M+H⁺] m/z 447.1236, calc for C₂₉H₁₉O₅; 447.1237.

Compounds 22 and 8 were characterized by UV-visible spectroscopy (FIG.16). Absorbance was measured using 15 μM solutions of each compound in asolution that was 90% aqueous, 10% DMSO. The peaks at 610 nm (lightdashed line) and 685 nm (light solid line) represent the anions ofcompounds 22 and 8, respectively, in NaOH (pH 12). The peaks at 605 nm(heavy dashed line) and 805 nm (heavy solid line) represent the neutralforms of compounds 22 and 8 at pH 7.4, respectively. Thus, the 3-1transposition resulted in red-shifted absorption spectra of about 75-200nm, depending on the ionization state.

It is interesting to note that for the transposed compound, ionizationof the hydroxyl leads to shorter wavelength absorption. This is counterto what is observed for the known regiochemistry. It is also apparentfrom these first two examples that the 3-1 transposition's effect onspectral properties (red shifted spectra) is more pronounced when thecarboxylate is replaced by a methyl ester.

The unique location of hydroxyl groups provided by the 3-1 transpositionleads to increased interaction with the xanthene oxygen. This is onepossible explanation for the observed red shift and unexpected behavior.Molecular modeling has demonstrated that in the neutral form, thehydroxyl proton of the transposed structure is held tightly with twohydrogen bonds and that in the anionic form, the xanthene oxygen is moreelectropositive for the transposed vs. the known regiochemistry(Mulliken charge of −0.289 vs. −0.386). Work is currently ongoing tofurther characterize this interaction and employ this strategy tofurther tune the spectral properties of xanthene-based structureswithout the need for complicated synthetic strategies which can includereplacing the xanthene oxygen with more electropositive elements (i.e.S, Se, C, Si, etc.). In recent work Nagano and co-workers have used asimilar strategy to extend the absorption and emission wavelength ofrhodamines and pyronines by approximately 80 nm each (DOI:10.1021/cb1002416). It should be noted that this approach retains therelatively small Stokes shifts (15-20 nm) present in the parentstructure. However, the transposition approach disclosed hereinincreased the Stokes shift. Additionally, the emission of compoundsreported by Nagano and co-workers are ca. 100 nm or more to the blue ofthose resulting from the 3-1 transposition approach described herein.

Compounds 22 and 8 also were characterized by their fluorescenceemission spectra. Fluorescence was measured using 15 μM solutions ofeach compound in a solution that was 90% aqueous, 10% DMSO For compound22, the longest wavelength emission observed from the anion occurrednear 690 nm at pH 9. For compound 8 (transposed), emission was notobserved. Given the greater than 800 nm absorbance peak and thetypically large Stokes shifts observed in this class of compounds, it islikely that the emission was beyond the working range of the instrument(>850 nm). Assuming that emission of the transposed compound is beyond850 nm, the 3-1 transposition resulted in red-shifted emission of morethan 160 nm.

The Stokes shift of each compound was measured and determined to beabout 80 nm for the compound 22 anion, and unmeasurable for the compound8 (transposed) neutral form. It is likely that the 3-1 transpositionalso enhanced the Stokes shift.

Absorbance and fluorescence of compounds 22 and 8 also were measured inmethanol. Compound 22 exhibited an absorption maximum at 598 nm.Compound 8 exhibited an absorption maximum at 701 nm. Compound 22exhibited an emission maximum at 688 nm. Compound 8 exhibited anemission maximum at 816 nm. Thus, placement of the hydroxyl groupproximal to the xanthene internal oxygen in compound 8 produced abathochromic shift of 103 nm in the absorption spectrum. Thetransposition also imparted a corresponding shift in the excitationspectrum, and a large bathochromic shift of 128 nm in the fluorescenceemission. Importantly, compound 8 absorbs and emits in the near-infraredwith absorption and emission maxima of 701 nm and 816 nm, respectively.Analogous trends were observed in other solvent systems, e.g., DMSO,DMSO:aqueous 1:1 (pH 9), DMSO: aqueous 1:9 (pH9), DMSO: aqueous 1:1 (pH12.1, NaOH), and DMSO: aqueous 1:9 (pH 12.1, NaOH).

Compounds 20a and 15a (type [c] annulated seminaphthofluorescein methylester with known regiochemistry versus annulated type [c]seminaphthofluorescein methyl ester with 3-1 transposition)

Some type [c] Seminaphthofluoresceins with the regiochemistry of theionizable group on the carbon 3 have been commercially available.However, given the large effect observed when the carboxylate wasreplaced with a methyl ester on compounds with the transposed geometryabove, the previously unreported seminaphthofluorescein methyl esteranalogue was prepared for direct comparison with new transposedcompounds.

Compound 20a was synthesized (Scheme 3) by condensing compound 17a (0.25g, 0.968 mmol) and compound 18a (0.232 g, 1.45 mmol) under acidicconditions. Compound 19a was isolated by flash column chromatography onsilica gel using EtOAc:MeOH 9:1 for elution. Yield 303 mg, 82%. Compound19a (50 mg, 131 μmol) was esterified as described previously forcompound 22. Compound 20a was isolated by flash column chromatography onsilica gel using EtOAc:MeOH 9:1 for elution. Yield 27 mg, 52%. ¹H NMR(600 MHz, DMSO) δ 10.57 (s, 1H), 8.53 (d, J=9.1 Hz, 1H), 8.25 (dd,J=8.0, 1.0 Hz, 1H), 7.90 (td, J=7.5, 1.3 Hz, 1H), 7.81 (td, J=7.8, 1.3Hz, 1H), 7.53 (dd, J=14.7, 7.7 Hz, 2H), 7.34 (dd, J=9.1, 2.4 Hz, 1H),7.23 (d, J=2.3 Hz, 1H), 6.86 (d, J=9.6 Hz, 1H), 6.78 (d, J=89 Hz, 1H),6.50 (D, J=2.0 Hz, 1H), 6.47 (dd, J=9.6, 2.0 Hz, 1H), 3.56 (s, 3H). ¹³CNMR (151 MHz, DMSO) δ 183.52, 165.24, 159.27, 158.26, 150.81, 149.36,137.52, 134.26, 133.27, 130.72, 130.69, 130.04, 192.92, 129.85, 129.48,124.47, 123.27, 122.90, 119.85, 117.45, 116.08, 113.91, 110.07, 104.55,52.10. HR ESI [M+H⁺] m/z 397.1068; calc for C₂₅H₁₇O₅; 397.1081.

Compound 15a was synthesized (Scheme 2) by condensing compound 12a(0.250 mg, 0918 mmol) and compound 13a (0.232 g, 1.45 mmol) under acidicconditions. Compound 14a was isolated by flash column chromatography onsilica gel using CH₂Cl₂:MeOH 9:1 for elution. Yield 300 mg, 81%.Compound 14a was esterified as described previously for compound 22.Compound 15a was isolated by flash column chromatography on silica gelusing CH₂Cl₂:MeOH 9:1 for elution. Yield 40 mg, 77%. ¹H NMR (600 MHz,DMSO) δ 10.78 (s, 1H), 8.25 (d, J=7.9 Hz, 1H), 7.91 (td, J=7.6, 1.3 Hz,1H), 7.86-7.77 (m, 1H), 7.63-7.51 (m, 3H), 7.40 (s, 1H), 7.13 (s, 1H),6.86 (d, J=9.7 Hz, 1H), 6.81 (d, J=8.7 Hz, 1H), 6.50 (dd, J=9.7, 1.9 Hz,1H), 6.34 (d, J=2.0 Hz, 1H), 3.55 (s, 3H). HR ESI [M+H⁺]m/z 397.1079;calc for C₂₅H₁₇O₅; 397.1081.

Compounds 20a and 15a were characterized by UV-visible spectroscopy(FIG. 17). Absorbance was measured using 15 μM solutions of eachcompound in a solution that was 90% aqueous, 10% DMSO. The peaks at 545nm (light dashed line) and 595 nm (light solid line) represent therespective anions, compounds 20c and 15h, in NaOH (pH 12). The peaks at515 nm (heavy dashed line) and 525 nm (heavy solid line) represent thepredominantly neutral forms of compounds 20a and 15a at pH 6,respectively. Thus, the 3-1 transposition resulted in red-shiftedabsorption spectra of about 10-55 nm, depending on the ionization state.

Compounds 20 and 15a also were characterized by their fluorescenceemission spectra. Fluorescence was measured using 15 μM solutions ofeach compound in a solution that was 90% aqueous, 10% DMSO. For compound20a, the longest wavelength emission observed from its correspondinganion, compound 20c, occurred near 640 nm in NaOH (pH 12). For compound15a (transposed), the longest wavelength emission observed from thecorresponding anion, compound 15h, occurred near 760 nm in NaOH. The 3-1transposition resulted in red-shifted emission by about 120 nm for thelongest wavelength-emitting species.

The Stokes shift of each compound was measured and determined to beabout 95 nm for the compound 20c anion, and about 165 nm for thecompound 15h (transposed) anion. Thus, the 3-1 transposition enhancedthe Stokes shift by about 70 nm.

Absorbance and fluorescence of compounds 20a and 15a also were measuredin methanol. Compound 20a exhibited absorption maxima at 487 nm and 521nm. Compound 15a exhibited absorption maxima at 501 nm and 536 nm.Compound 20a exhibited an emission maximum at 550 nm. Compound 15aexhibited an emission maximum at 582 nm. Thus, placement of the hydroxylgroup proximal to the xanthene internal oxygen in compound 15a produceda bathochromic shift of 15 nm in the absorbance spectrum. Thetransposition also imparted a corresponding shift in the excitationspectrum, and a large bathochromic shift of 32 nm in the fluorescenceemission.

Although the red shift of compound 15a in methanol was modest, itscorresponding anion (compoundl5h) in DMSO:aqueous base 1:9 was shiftedmore substantially by 50 nm to 599 nm as compared to anion 20c under thesame conditions. Its fluorescence emission was shifted by an even larger130 nm, emitting at 760 nm. The comparatively large shift of the anion15h supports a polar effect as playing a key role in modulating theHOMO-LUMO gap in the compound. The oxoanion of asymmetric 15h isexpected to have a more significant field effect on the polarity of theproximal ether oxygen as compared to the corresponding oxoanion 20c.Like many other NIR-emitting compounds, compound 15h displays arelatively low quantum yield (less than 1%) in aqueous solution. Thebrightness of compound 15h is comparable to other NIR probes.

Compounds 15b and 15d (type [c] annulated seminaphthorhodafluor methylesters (free amino and dimethyl, respectively) with 3-1 transposedregiochemistries)

The methyl ester analogue of known seminaphthofluorescein behavessimilarly to previously reported compounds. Only a slight red shift, onthe order of approximately 10 nm as compared to the carboxylatecontaining compound (Whitaker et al. Analytical Biochemistry, 1991, 194,330-344), was observed. Thus the methyl ester analogues of knownseminaphthorhodafluors (alternatively known as seminaphthorhodols) werenot prepared for direct comparison. Literature values for similarcompounds with the known regiochemistry were used for comparison withcompounds 15b and 15d.

Compound 15b (free amino) was characterized by UV-visible spectroscopy(FIG. 18), and compared to C.SNARF-5 (literature,SNARF=seminaphthorhodafluor). Absorbance was measured using a 15 μMsolution of compound 15b in a solution that was 90% aqueous, 10% DMSO.The peak at 535 nm (heavy line) represents the predominantly phenolicform of 15b at pH 6. The peak at 615 nm (light line) represents thephenoxide of compound 15b at in NaOH (pH12). In comparison, C.SNARF-5produces a peak at 550 nm (anionic form) and a peak at 485 nm (neutralform) (Whitaker et al., Anal. Biochem. 1991, 194, 330-344). Thus, the3-1 transposition resulted in red-shifted absorption spectra of about50-65 nm, depending on the ionization state.

Compound 15b also was characterized by its fluorescence emissionspectrum and compared to C.SNARF-5. Fluorescence was measured using a 15μM solution of compound 15b in a solution that was 90% aqueous, 10%DMSO. For C.SNARF-5, the longest wavelength emission observed from theanion occurred near 632 nm (Whitaker et al., Anal. Biochem. 1991, 194,330-344). For compound 15b (transposed), the longest wavelength emissionobserved from the phenoxide occurred near 770 nm. The 3-1 transpositionresulted in red-shifted emission by about 138 nm for the longestwavelength-emitting species.

The Stokes shift of each compound was measured and determined to beabout 82 nm for the C.SNARF-5 anion, and about 155 nm for the compound15b (transposed) phenoxide. Thus, the 3-1 transposition enhanced theStokes shift by about 73 nm.

Compound 15d (dimethyl) was characterized by UV-visible spectroscopy(FIG. 19), and compared to SNARF-1 (literature). Absorbance was measuredusing 15 μM solutions of compound 15d in a solution that was 90%aqueous, 10% DMSO. The peak at 570 nm (heavy line) represents thepredominantly neutral form of 15d at pH 6. The peak at 630 nm (lightline) represents the phenoxide of compound 15d in NaOH (pH 12). Incomparison, SNARF-1 produces a peak at 573 nm (anionic form) and a peakat 515 nm (neutral form) (Whitaker et al., Anal. Biochem. 1991, 194,330-344). Thus, the 3-1 transposition resulted in red-shifted absorptionspectra of about 55-57 nm, depending on the ionization state.

Compound 15d also was characterized by its fluorescence emissionspectrum and compared to SNARF-1. Fluorescence was measured using a 15μM solution of compound 15d in a solution that was 90% aqueous, 10%DMSO. For SNARF-1, the longest wavelength emission observed from theanion occurred near 631 nm (Whitaker et al., Anal. Biochem. 1991, 194,330-344). For compound 15d (transposed), the longest wavelength emissionobserved from the phenoxide occurred near 780 nm (NaOH, pH 12). The 3-1transposition resulted in red-shifted emission by about 149 nm for thelongest wavelength-emitting species.

The Stokes shift of each compound was measured and determined to beabout 58 nm for the SNARF-1 anion, and about 150 nm for the compound 15d(transposed) phenoxide. Thus, the 3-1 transposition enhanced the Stokesshift by about 92 nm.

Compounds 20b and 15g (type [c] annulated seminaphthorhodamine methylesters (free amino) with known and 3-1 transposed regiochemistries,respectively)

To the best of the inventors' knowledge, the full seminaphthorhodamineanalog with type [c] annulation and the known regiochemistry has notbeen reported. We prepared this compound (20b) for direct comparisonwith a new seminaphthorhodamine with the 3-1 transposition (15g).

Compounds 20b and 15g were characterized by UV-visible spectroscopy(FIG. 20). Absorbance was measured using 15 μM solutions of eachcompound in a solution that was 90% aqueous, 10% DMSO. The peaks at 540nm (dashed line) and 570 nm (solid line) represent the cations ofcompounds 20b and 15g, respectively, at pH 7.4. Thus, the 3-1transposition resulted in a red-shifted absorption spectrum of about 30nm.

Compounds 20b and 15g also were characterized by their fluorescenceemission spectra. Fluorescence was measured using 15 μM solutions ofeach compound in a solution that was 90% aqueous, 10% DMSO. For compound20b, the longest wavelength emission observed from the cation occurrednear 640 nm at pH 7.4. For compound 15g (transposed), the longestwavelength emission observed from the cation occurred near 770 nm at pH7.4. The 3-1 transposition resulted in red-shifted emission by about 130nm.

The Stokes shift of each compound was measured and determined to beabout 100 nm for the compound 20b cation, and about 200 nm for thecompound 15g (transposed) cation. Thus, the 3-1 transposition enhancedthe Stokes shift by about 100 nm.

Compounds 15e and 15f (Type [c] annulated Seminaphthorhodafluor methylesters (free amino and dimethyl) with 3-1 transposed regiochemistriesand the ionizable moieties transposed)

In addition to the previous examples, a second series ofseminaphthorhodafluors (15e-15f) with the 3-1 transposition was preparedin which the ionizable hydroxyl and amine (free amino and dimethyl)functionalities also were transposed. To the best of the inventors'knowledge, no directly comparable structure has been reported in theliterature. The nearest compound for comparison, a type [c] annulatedseminaphthorhodafluor with the known regiochemistry, was reported to“display longer wavelength fluorescence, emitting at 600 nm in MeOH whenexcited at 525 nm.” (Clark et al., Tetrahedron Lett., 2004, 45,7129-7131.)

The new series of compounds based on transposition of the ionizablehydroxyl and amine functionalities combined with the 3-1 transposedregiochemistry disclosed herein displays unanticipated acid-baseproperties. Known seminaphthorhodafluors display long wavelengthabsorption at high pH; however, the free amino compound in this seriesrespond to low pH with increased long wavelength absorption. FIG. 21shows a series of pH titration curves of compound 15e, demonstratingincreased long wavelength absorption and decreased short wavelengthabsorption at low pH.

Preliminary data also indicate that this series (free amino anddimethyl) exhibit red-shifted spectra comparable to others compoundswith the 3-1 transposition disclosed herein. Emission maxima of thelongest wavelength emitting species are in the range of ˜740-780 nm. Itis apparent the red-shifted compounds based on the combination oftranspositions are considerably more red shifted as compared to thepreviously reported compounds.

Compound10-7-(2,5-dimethylphenyl)-13-hydroxy-1H-dibenzo[c,h]xanthen-1-one

1,8-dihydroxynaphthalene (0.298 mg, 1.86 mmol) and2,5-dimethyl-benzaldehyde were suspended in 2 mL of 85% H₃PO_(4,) themixture was vigorously stirred and heated at 125° C. for 24 hours. Themixture was allowed to cool down to room temperature and then pouredinto 50 mL of water. The precipitate formed was filtered and washed withwater (2×50 mL). The target compound is isolated by flash columnchromatography on silica gel using CH₂Cl_(2:MeOH) 9:1 for elution. Yield67 mg, 17%. ¹H NMR (400 MHz, CDCl₃) δ 7.59-7.45 (m, 4H), 7.35 (s, 2H),7.16 (s, 2H), 7.10 (s, 2H), 7.04 (d, J=6.8 Hz, 2H), 6.94 (d, J=8.9 Hz,2H), 2.45 (s, 3H), 2.00 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 155.96,138.17, 136.37, 135.49, 133.06, 132.79, 130.87, 130.65, 129.51, 121.04,120.73, 120.52, 117.21, 113.46, 21.18, 19.43. HR ESI [M+H₊] m/z417.1476, calc for C₂₉H₂₁O₃; 417.1496.

Compound 16a-methyl2-(1-methoxy-10-oxo-10H-benzo[c]xanthen-7-yl)benzoate

Under argon atmosphere, compound 14a (0.050 g, 0.131 mmol) and K₂CO₃(0.072 g, 0.523 mmol) were suspended in 600 μL of anhydrous DMF. CH₃I(0.111 g, 0.785 mmol) was added in one portion and the mixture heated at60° C. for 24 hours. The mixture was allowed to cool down to roomtemperature and then 5 mL of saturated NH₄Cl aqueous solution was added.Compound 16a was isolated by flash column chromatography on silica gelusing CH₂Cl₂:MeOH 9:1 for elution. Yield 9.8 mg, 18%. ¹H NMR (400 MHz,CDCl₃) δ 8.29 (dd, J=7.8, 1.1 Hz, 1H), 7.80-7.73 (m, 1H), 7.74-7.67 (m,1H), 7.60 (t, J=8.0 Hz, 1H), 7.47 (d, J=8.9 Hz, 1H), 7.42 (d, J=7.6 Hz,1H), 7.35 (dd, J=7.5, 1.0 Hz, 1H), 7.07 (d, J=7.5 Hz, 1H), 6.93 (dd,J=9.1, 5.3 Hz, 2H), 6.68 (q, J=1.8 Hz, 2H), 4.13 (s, 3H), 3.59 (s, 3H).¹³C NMR (101 MHz, CDCl₃) δ 185.38, 165.66, 158.82, 158.40, 151.35,150.61, 137.97, 135.12, 132.78, 131.21, 130.67, 130.38, 130.32, 129.67,129.27, 124.52, 123.41, 120.49, 118.58, 116.77, 114.92, 107.91, 105.48,56.26, 52.39. HR ESI [M+H₊] m/z 411.1231; calc for C₂₆H₁₉O₅; 411.1237.

Methylation at R¹⁸ to produce methyl ether 16a dramatically improved thequantum yield. The quantum yield of yellow-orange emitting 16a was0.4649, 40 times greater than the corresponding neutral compound 15a. Itwas slightly higher than the yellow-green emitting neutral compound 20a,and >2 times higher than orange-red emitting anion 20c. The compounds(7.5 μM) were analyzed in 10:90 DMSO:aqueous solutions. Fluorescenceemission was readily visible in a darkened room when the solutions wereexcited from below with 3-watt megaMAX 505 nm ALS system. Theemission-enhancing properties afforded through the combination of the3-1 transposition and methylation is in contrast to previously reportedmethyl ethers of seminaphthofluorescein compounds.

The interested reader is referred to online resources atpubs.acs.org/doi/supp1/10.1021/ja302445w/supp1_file/ja302445w_si_001.pdffor additional NMR, ESI, and absorption spectra, as well as HOMO-LUMOsurfaces of some embodiments of the disclosed compounds.

Example 2 Emission in Blood

Compound 15d (Table 1) was diluted to 40 μM in 50% DMSO:50% 25 mM pH 9phosphate buffer. As shown in FIG. 22, the diluted compound fluorescedwith λ_(max ex)=˜690 nm and λ_(max em)=˜790 nm (Stokes shift=˜100 nm,1834 cm⁻¹). Inclusion of whole blood (porcine blood in Na-EDTA, LampireBiological Laboratories) in the aqueous portion of this solvent toproduce a final blood concentration of 5% by volume resulted in onlyminimal loss of fluorescence attributed to hemoglobin absorption andscattering from blood components. Emission remained stable for at least1 hr. The absorption profile matches the output of a laser pointer(635-670 nm; max output <5 mW). NIR emission (80 μM) was clearly visiblewith a digital camera (Kodak EasyShare Z740 in a NIR photograph takenwith sec. exposure collected through a Hoya R72 camera filter (%T<1% at<690 nm, %T>90% at >750 nm). Similar results were observed with 5% wholeblood, but with increased scattered excitation light clearly visible inthe both visible and NIR images, an issue that was easily overcome witha second filter.

The close match between compound 15d's absorption and the output of acommon laser pointer allowed its excitation with a simple inexpensivelight source.

Furthermore, its NIR emission was detected with a commercial-gradedigital camera in normal room light combined with an inexpensive HoyaR72 infrared filter (FIG. 22 inset).

The absorption of compound 4a (Table 1) was clearly visible and remainedstable over extended periods of time in the blood solution. Compound 5a(Table 2) was also found to function in 5% blood. Its absorbance andfluorescence spectra faded over time, but remained visible after aboutan hour (although with only 10-20% of its original emission intensity).Molecular modeling has shown favorable structural interactions ofcompounds 5f and 24 with glutathione (FIG. 12—compound 24).

Spectral behavior of compound 15g (type [c] annulatedseminaphtho-rhodamine methyl ester (free amine) with the 3-1transposition) was investigated to determine its potential for sensinguse in whole blood. Solutions of compound 15g in 10% or 90% by volumewhole blood (porcine blood in Na-EDTA, Lampire Biological Laboratories)were investigated in a 3×3 mm cell. The fluorophore was first dissolvedin 50 mM, pH 7.4 phosphate buffer to ensure complete dissolution, and inthe case of 90% whole blood, this stock solution was mixed with wholeblood in a 1:9 ratio. In the case of 10% whole blood, 1 part whole bloodwas added to 9 parts of an appropriately diluted (with deionized water)solution of compound 15g stock. In both cases, the final concentrationof compound 15g was 150 μM with a final phosphate buffer concentrationof 5 mM. FIG. 23A shows the absorbance spectra of compound 15g in 10%whole blood compared to 10% whole blood without the compound. Throughoutmuch of the spectrum, the dye produces a greater absorbance than theblood alone. The change in absorbance between the two spectra providesthe effective absorbance of the dye. As shown in FIG. 23A, there is arelatively high transparency blood “window” extending from about 650 nmto 850 nm. The broad absorption and large Stokes shift of this dyeallows significant excitation with greater than 50% efficiency atwavelengths beyond that of the major hemoglobin peaks in blood. FIG. 23Bshows the emission spectra of compound 15g in 10% and 90% whole bloodsamples upon excitation at 670 nm. As shown in FIG. 23B, dye emission isclearly seen in both blood samples with an emission spectrum maximum ofabout 750 nm. The photograph inset in FIG. 23B demonstrates that 10 and90% whole blood samples (left and right, respectively) are nearlyindistinguishable by the naked eye.

Compound 8 (type [c] fully annulated naphthofluorescein methyl esterwith 3-1 transposition) displays a maximum absorption at greater than800 nm, well past that of hemoglobin absorption in blood. As a result,compound 8 also may perform well in blood.

Example 3 Synthesis and Characterization of Rhodamine Bis-Boronic Acids

Compound 51—seminaphthorhodamine bis-boronic acid: With reference toScheme 9 (FIG. 10), the starting material,2-(4-amino-2-hydroxybenzoyl)benzoic acid was obtained via the basichydrolysis of rhodamine 110 at 160° C. over 3 hours in 92% yield. Acidcondensation with 1,6-aminohydroxynaphthalene using a 1:1 mixture ofCH₃SO₃H:TFA afforded the corresponding seminaphthorhodamine 50 in 63%yield after isolation by flash column chromatography with CH₂Cl₂:MeOH,9:1. Reductive amination to produce the corresponding bis-boronic acid51 was carried out in dichloroethane (DCE) in two steps. Other solventsincluding MeCN and THF were also used, but gave low yields (less than1%) of the desired target. The first step involved the reaction of 50with 2-formylboronic acid and triacetoxy sodium borohydride undermicrowave irradiation at 130° C. for 40 minutes. The microwave vial wasopened, acetic acid was added in one portion, and the mixture was heatedfor an additional 40 min at 130° C. The mixture was neutralized withsaturated NaHCO_(3,) and the target compound was isolated by preparativeTLC using CH₂Cl₂:MeOH 9:1. The target compound was characterized by ¹HNMR, HR ESI MS, and its purity (96%) was determined by reversed phaseHPLC.

Compound 53—naphthorhodamine bis-boronic acid: With reference to Scheme10 (FIG. 11), naphthorhodamine 52 was synthesized in 74% yield by theacid promoted condensation of phthalic anhydride and1,6-aminohydroxy-naphthalene via heating in the presence oftrifluoromethane sulfonic acid at 100° C. for 2 hours, then at 140° C.for an additional 2 h under argon. Reductive amination to produce thecorresponding bis-boronic acid 53 was carried out in DCE in two steps asdescribed above. Optimal reaction conditions were obtained by monitoringthe reaction by reversed phase HPLC. The target compound was isolated in38% yield by preparative TLC using CH₂Cl₂:MeOH 95:5 for elution.Naphthorhodamine 52 and the target compound were characterized by ¹HNMR, HR ESI MS, and their purity (99% and 95% respectively) wasdetermined by reversed phase HPLC on a C₁₈ reversed phase column and agradient solvent system composed of H₂O:1% TFA in MeCN for elution.

Characterization: The spectral properties of compounds 50-53 weredetermined. Absorption and fluorescence spectra of solutions of therhodamine bis-boronic acids 51 and 53, and their respective precursors50 and 52, are shown in FIGS. 27A-C (in DMSO:buffer 1:9) and 27D-F(DMSO:buffer 1:1). Precursor 50 (2.25 μM) and bis-boronic acid compound51 (3.75 μM) were excited and monitored at 500 nm and 675 nm. Precursor52 (12.5 μM) and bis-boronic acid compound 53 (7.5 μM) were excited andmonitored at 590 nm and 700 nm. Excitation and emission spectra werenormalized to their absorbance at the excitation wavelength, and areproportional to quantum yield.

The results are summarized below in Table 5. The seminapthorhodamine 50maximum absorption in DMSO buffer 1:9 was at 535 nm (40 nm to the red ofrhodamine 110). Its maximum emission at 628 nm displayed a reasonablequantum yield of 19% and was red shifted nearly 100 nm as compared torhodamine 110. The corresponding bis-boronic acid 51 was further redshifted with maximum absorption and emission at 560 and 638 nm,respectively. Emission was slightly quenched through photoinducedelectron transfer (PET) in the boron-nitrogen system as evidenced by thelower quantum yield. It is interesting to note that the quenching forthe original “RhoBo” and the fully annulated bis-boronic acid 53discussed below is much greater than the asymmetric bis-boronic acid 51.

The maximum absorption of napthorhodamine 52 was further shifted to 578nm with emission wavelength and quantum yield (668 nm, 10%) comparableto commercially available naphthofluorescein. The correspondingbis-boronic acid 53 was further red shifted with maximum absorption andemission at 628 and 692 nm, respectively. Although it displayed areasonably strong blue/green color, its fluorescence was nearlycompletely quenched, allowing for a potential turn-on type sensor. Likethe other compounds, its emission was slightly redder and its quantumyield was slightly higher in DMSO buffer 1:1. However, like itscorresponding rhodamine precursor 52, it exists primarily in the closedand colorless lactone form in this solvent.

TABLE 5 Abs DMSO:buffer Compound (ε, M⁻¹cm⁻¹) Ex/Em (Q.Y.) Brightnessratio^(a) Rhodamine 110^(b) 495 (66800 @ 492) NA/523 (0.91) 60788 @ 492NA RhoBo 501 (22149) 502/528 (0.30)^(c,f) 6645 1:9 50 535 (42701)534/628 (0.19) 8083 1:9 Bis-boronic acid 51 560 (20326) 568/638(0.15)^(e) 3032^(e) 1:9 52 578 (11994)^(d) 584/668 (0.10) 1144^(d) 1:9Bis-boronic acid 53 628 (10759)^(d) 612/692 (0.004)^(f)  43^(d,f) 1:9Rhodamine 110 503 (67905) 502/526 (NA) NA 1:1 RhoBo 504 (21003) 504/528(NA) NA 1:1 50 542 (21064) 542/630 (0.30) 6298 1:1 Bis-boronic acid 51579 (23096) 580/643 (0.35) 8037 1:1 52 602 (1081)^(g) 596/676 (0.11) 121^(g) 1:1 Bis-boronic acid 53 616 (1616)^(g) 624/700 (0.18)  292^(g)1:1 ^(a)Final pH 7.4 phosphate buffer concentration was 12.5 mM.^(b)Solvent was 10 mM HEPES, pH 7.5, 15% (v/v) EtOH; Values from Leytuset al., Biochem. J. 209, 299 (*1983). ^(c)Value taken from Halo et al.,J. Am. Chem. Soc. 131, 438 (2008). ^(d)Partial lactone formation.^(e)Weak PET quenching, ^(f)Strong PET quenching, ^(g)Near completelactone formation.

Example 4 Saccharide Sensing and Selectivity

Responses of the boronic acid probes, compounds 51 and 53, weremonitored over a wide range of glucose, ribose, and fructoseconcentrations in various solvents. Solvents including MeCN, MeOH, EtOH,DMSO and buffer were initially screened for sugar sensing. Probes werepartially soluble in MeCN and buffer, and soluble in MeOH, EtOH andDMSO. DMSO mixtures were chosen for further studies.

Due to the solvent-dependent lactone ring opening-closing equilibrium(Scheme 11) that these type of probes exhibit, a DMSO titration wascarried out, in order to determine the range of possible conditions forsugar sensing. Samples were titrated from 0-60% DMSO. The final pH 7.4phosphate buffer concentration was 12.5 mM. Absorbance values weremeasured as follows: seminaphthorhodamine 50—532 nm, bis-boronic acid51—578 nm, naphthorhodamine 52—578 nm, bis-boronic acid 53—627 nm. Asshown in FIG. 28, at lower DMSO concentration, rhodamine precursors 50and 52 as well as the bis-boronic acids 51 and 53 exist at least to someextent as the colored carboxylate species. As the DMSO concentrationincreased, the lactone form predominated (low absorbance values) for allbut bis-boronic acid 51. For this case, the carboxylate formpredominated over this DMSO concentration range. At DMSO concentrationsof 60-90%, precipitation occurred for all cases; however, at 90% DMSO,all compounds were soluble and predominantly in their colorless closedlactone forms.

It is interesting to note that the boronic acid derivatives are lessprone to lactone formation than their corresponding rhodamineprecursors.

Initial screening for sensing of glucose, fructose and ribose usingthese probes in mixtures DMSO:buffer 9:1 (final pH 7.4 phosphate bufferconcentration of 12.5 mM) used in our previous work, showed that nochanges in either absorbance or fluorescence were observable. Incubationeither at room temperature or 37° C. for up to 24 h, gave similarresults. It appears that sugar binding did not sufficiently alter thelactone equilibria in this solvent.

The spectral behavior of both compounds 51 and 53 in the presence ofsaccharides was further investigated in both 1:1 and 1:9 DMSO-buffer(final pH 7.4 phosphate buffer concentration of 12.5 mM) solutions. Bothbis-boronic acids responded to binding of sugars through a red shift intheir emission and the expected increase in fluorescence intensity.

FIGS. 29A-D show the response of bis-boronic acid 51 to fructose, riboseand glucose under the two solvent conditions mentioned above. FIG. 29Ais absorption and emission spectra of compound 51 (3.75 μM) in responseto 10 mM sugars in DMSO:buffer 1:9; FIG. 29B is fluorescence emission(ex. 580 nm/em. 660 nm) as a function of sugar concentration inDMSO:buffer 1:9; FIG. 29C is absorption and emission spectra of compound51 (3.75 μM) in response to 10 mM sugars in DMSO:buffer 1:1; FIG. 29D isfluorescence emission (ex. 580 nm/em. 640 nm) as a function of sugarconcentration in DMSO:buffer 1:1. The final pH 7.4 phosphate bufferconcentration was 12.5 mM. The horizontal lines in FIGS. 29B, 29Drepresent fluorescence of compound 51 in the absence of analyte. In the1:9 DMSO:buffer system, this asymmetric rhodamine bis-boronic acidresponded more strongly to fructose (FIGS. 29A and 29B). An increase influorescence intensity was observed for fructose and ribose at thelowest concentration investigated. At high sugar concentrations, theresponse for all three sugars converged. A near 2-fold increase inabsorbance resulted in a greater than 5-fold increase in fluorescencefor all sugars. The solvent system was found to have a large effect onthe response. Bis-boronic acid 51 responded most strongly to ribose inthe 1:1 DMSO buffer system (FIGS. 29C and 29D). An approximately1.5-fold increase in both absorbance and fluorescence was observed overthe entire concentration range investigated. Binding of all three sugarsoccurred at lower concentrations as compared to the 1:9 DMSO:buffersystem, with the response of both ribose and fructose having alreadyplateaued at 10 mM, the lowest concentration investigated. Unlike the1:9 solvent system, there were significant spectral differences in theresponses toward different sugars (FIG. 29C compared to FIG. 29A).

There were significant differences between the responses of asymmetricbis-boronic acid 51 and symmetric bis-boronic acid 53. FIGS. 30A-D showthe response of compound 53 to fructose, ribose and glucose in both 1:9and 1:1 DMSO:buffer systems. FIG. 30A is absorption (630 nm) spectra ofcompound 53 (7.5 μM) as a function of sugar concentration in DMSO:buffer1:9; FIG. 30B is fluorescence (ex. 630/em. 690 nm) spectra of compound53 (7.5 μM) as a function of sugar concentration in DMSO:buffer 1:9;FIG. 30C is absorption (640 nm) spectra of compound 53 (7.5 μM) as afunction of sugar concentration in DMSO:buffer 1:1; FIG. 30D isfluorescence (ex. 640/em. 700 nm) spectra of compound 53 (7.5 μM) as afunction of sugar concentration in DMSO:buffer 1:1; The final pH 7.4phosphate buffer concentration was 12.5 mM. Horizontal lines representabsorbance and fluorescence of compound 53 in the absence of analyte. Ofthe three sugars screened, compound 53 responded almost exclusively tofructose at concentrations below 100 mM in the 1:9 DMSO-buffer system(FIGS. 30A, 30B). At the highest concentration of fructose investigated,a less than 3-fold increase in absorbance at 630 nm resulted in agreater than 140-fold turn-on response of fluorescence emission at 710nm upon excitation at 630 nm. No spectral shifts were observed inresponse to any of the sugars. The increase in absorbance may be atleast partially the result of lactone opening upon sugar binding. Theincrease in fluorescence is largely the result of near completedisruption of the PET quenching in the boron-nitrogen system of the freerhodamine bis-boronic acid. It was estimated that the quantum yield ofcompound 53 after binding approaches 20% or more (140-fold fluorescenceincrease divided by 3-fold absorbance increase multiplied by the quantumyield of the free compound 53=0.186) which is greater than the quantumyield of precursor 52. Secondary interactions and additionalrigidification of the chromophore upon binding could be responsible forthis further enhancement of emission. Again, the solvent system wasfound to have a major effect. Affinity for ribose was greatly increased,approaching the response observed for fructose at the lowestconcentration investigated (10 mM) upon changing the solvent system to1:1 DMSO-buffer. Binding of all three sugars at lower concentrations(FIGS. 30C and 30D) was found to increase, and the response of all threesugars converged to nearly a 5-fold increase in emission at 720 nm uponexcitation at 640 nm. Free bis-boronic acid 53 in 1:1 DMSO-buffer wascolorless but binding of sugars promoted opening of the lactone leadingto a green-blue color.

When the solvent system is mostly aqueous (1:9 DMSO:buffer), solvationby water inhibits intramolecular interactions by salt bridges, allowingthe sugar boronate complexes in compound 51 to adopt practically anypossible conformation reaching the carboxyl group without problem,especially for the side where the rhodamine system ring is not extended.For the relatively less aqueous system (1:1 DMSO:buffer), theselectivity turns slightly towards ribose due to the enhancement ofintramolecular binding interactions between the bound sugar and dye.FIG. 31 shows a model of ribose bis-boronate of compound 51 describingthe electrostatic interactions within the N—H—O—B atoms in a 1:1DMSO:buffer solution. Electrostatic attraction between N—H—O—B atomsrestricts rotation of the C—B bond resulting in selectivity for theribose complex. Only one of the boronates can reach the carboxylate.

Compound 53 has both boronic acid groups far from each other and fromthe carboxylate compared to compound 51. In the 1:9 DMSO:buffer solventsystem the selectivity followed the normal behavior(fructose>ribose>glucose). When the ratio DMSO:buffer is 1:9, theintramolecular electrostatic interactions are practically nonexistent,allowing more freedom to adopt many possible conformations. Ifsaccharide bis-boronates are formed, the interactions between them canbend the planar chromophore slightly to allow interactions with thecarboxylate. Upon changing the solvent system to 1:1 DMSO-buffer, theaffinity of symmetric compound 53 for ribose was greatly increased,approaching the response observed for fructose at relatively lowconcentrations. In this more organic solvent system, the asymmetricboronic acid compound 51 reversed its selectivity and responded moststrongly to ribose. For the system with DMSO:buffer 1:1 ratio, theelectrostatic interactions are very weak, but strong enough to restrictthe molecule from adopting many possible conformations. FIG. 32 shows amodel of a bis-boronate complex of compound 53 with two molecules offructose.

Both compounds 51 and 53 exhibited selectivity trends(fructose >ribose >glucose) in the 1:9 DMSO:buffer system. Compound 53is particularly attractive as a candidate for selective sugardetermination as it responded exclusively to fructose through a clear toblue-green color change with corresponding turn-on NIR fluorescenceenhancement of up to 140-fold and no interference from other sugars atconcentrations below 100 mM.

Example 5 Screening Dyes

Embodiments of the disclosed NIR dyes can be screened by determiningproperties of each fluorophore, including : 1) wavelength of maximumabsorption of each form (i.e., neutral and ionized); 2) molarabsorptivity of each form; 3) wavelength of maximum excitation of eachform; 4) wavelength of maximum emission of each form (corrected for pmt(photomultiplier) response); 5) Stokes shift in units of both nm andenergy of each form; 6) complete Excitation-Emission Matrix, EEM(corrected for pmt response) covering a wavelength region that includessolvent adduct, neutral and ionized forms (excitation 335-800 nm;emission 360-850 nm); 7) relative quantum yields of each form; and/or 8)pK_(a) (or apparent pK_(a) dependent on the solvent).

A standardized procedure including absorption spectra, excitationemission matrices (EEMs), emission spectra upon common “laser-line”excitations, and excitation spectra of the observed emission peaks,and/or serial dilutions of a pH titration series will provide the datarequired to characterize the dyes and their various forms.

The screening can be divided into two steps. The first step would be aninitial screen of compounds comprised of:

Step 1-A: A single absorption spectra and EEM of ˜7.5 M each compound(concentration will be lowered if the absorption is greater than about0.25, or increased if the absorption is less than about 0.05) in pH 8.25buffer with 5% DMSO (pH 8.25 is near or slightly greater than the likelypK_(a) of most of the dyes (with the exception of the proposedfluorinated derivatives), so both anionic and neutral forms will bepresent in significant (nearly equal) amounts). The DMSO will help withany potential solubility issues of the neutral form (the DMSO amount canbe reduced if the dye is sufficiently soluble).

Step 1-B: The same procedure as 1-A, but with 5% buffer, 95% DMSO.

Step 2: Multiple concentrations of the dyes (for molar absorptivity andquantum yield measurements) and multiple pHs (for pK_(a) measurements)will be investigated for the most promising candidates. When available,appropriate quantum yield standards will allow estimation of absolutequantum yields.

Step 3: Computer-aided molecular modeling can be employed to investigatepotential interactions with analytes of interest provided by the variousgeometries of the analytes and the dye molecules.

Interpretation of results. Steps 1A and 1B will provide a wealth ofpreliminary data (absorption and emission maxima of each species in bothsolvents, and an estimate of the relative quantum yield of the longwavelength species in both solvents). This initial first screen will notgive any information about molar absorptivity, pK_(a), or absolutequantum yields. Selected compounds can be investigated further in Steps2 and/or 3.

Example 6 Detecting and Quantitating Analytes in Biological Fluids

Promising dyes can be investigated to determine their binding withanalytes of interest (e.g., GSH, cysteine, homocysteine, SAICAr, S-Ado)and to determine any potential interferences from blood and/or urine. Aknown procedure used to quantify binding of albumin dyes to bloodcomponents can be utilized (Omoefe et al., J. of Biomed. Optics July2001, 6(3), 359-365).

Measurements can be made in artificial blood or urine solutionscontaining the major fluid components and spiked with an analyte ofinterest. The major blood components and their mean concentration (mg/L)in blood are: HSA—41,000, HDL—850, LDL—810, globulin—32,000, red bloodcells (43.5% hematocrit) 489,375 (Abugo et al., J. Biomed. Opt., 2001,6, 359-365; Abugo et al., Anal. Biochem., 2000, 279, 142-150). Thechemical composition of human urine has been thoroughly investigated andreported by NASA (NASA Contractor Report CR-1802). The composition ofsimulated urine and urine solutions will be based on their findings.Five major components (urea, creatinine, oxalate, uric acid and citrate)have previously been used to simulate urine content (Ow et al. IFMBEProceedings, 2008, Vol. 21, Part 3, Part 11, 742-745; Osman et al.,Biomed 2008 Proceedings 2008, 21, 742-745).

Apparent binding constants can be estimated directly from raw titrationdata without any model assumptions; however, the analyticalrepresentation of data may be used to simulate dye behavior in a complexmixture with multiple potential interferences. A phenomenologicalapproach involving fits of experimental titration curves may be usedmodel the experimental data and allow rough estimates of the binding ofvarious dyes in mixtures containing components of biological fluids. Itmay not be possible to test all blood (or urine) components; however,inclusion of the major components should be sufficient to screenembodiments of the disclosed dyes for selectivity toward analytes ofinterest. Matrix effects may be evaluated by comparing standardcalibration curves in the biological fluids with standard calibrationcurves in an optimal buffer system.

Selectivity may be fine-tuned based on the experimental results combinedwith further computer aided molecular simulations. Analysis of GSH andSAICAr and S-Ado may be carried out in authentic biological fluids (GSHin blood and SAICAr and S-Ado in urine) using samples spiked withcalibration (reference) standards and using quality control (QC)samples. The developed methodology may be evaluated according tostandard procedures for method development including (a) accuracy, (b)precision, (c) selectivity, (d) sensitivity, (e) reproducibility, and(f) stability. Recovery of analyte can be determined using standardaddition protocols. All biological fluids to be used may be purchasedfrom commercial sources.

Results from optimal conditions for quantification of the analytes ofinterest in solution along with a similar methodology as described byOrfanos (Anal. Biochem., 1980, 104, 70-74) may be adapted for thedetection and quantitation of the analytes of interest in biologicalfluids deposited in filter paper. Dried blood (or urine) specimens onfilter paper are obtainable. For blood spots, disks may be punched fromthe paper and the blood contents typically eluted/extracted into a smallvolume of solvent. In conventional analyses, the absorption fromhemoglobin remains a major interference. This is typically overcome byfurther dilution in order to ensure the hemoglobin content is less than30 μg/ml. Such dilution complicates measurements of analytes present athigh concentration and may even prevent analysis of trace analytes.Based on preliminary results (see Example 2), embodiments of thedisclosed NIR dyes will function in the presence of at least 5% wholeblood. Thus, extraction of the contents of a typical 4.8-mm disk into asmall volume (0.6 mL or less, containing one of our dyes) may be useddirectly without dilution.

In some instances, the dyes may be conjugated to a polymer, e.g., apolyethylene glycol, to provide enhanced selectivity and minimization ofinterferences. Fluorination of certain embodiments of the disclosed dyesmay facilitate solubility in aqueous solutions. In certain instances,dithiothreitol (DTT) may be added to the analysis solution to preventand/or minimize disulfide formation. At least some embodiments of thedisclosed dyes function in the presence of DTT; furthermore, monitoringtotal GSH-GSSG (via adding DTT to the solution) is feasible since about90% of blood GSH is in the reduced form and glutathione disulfide (GSSG)levels are similar in both patients and controls (Atkuri et al., PNASU.S.A., 2009, 106, 3941-3945).

These studies may enable identification of NIR dyes that bind stronglyand selectively to analytes of interest. Fluorescence enhancement uponbinding may be evaluated and utilized to determine what dye geometriesand functional groups favor target analytes or specific interferingcomponents.

Example 7 Glutathione Selectivity Evaluation

Mechanistic studies may be conducted in control experiments (bufferedmedia at physiological conditions or in the presence of a co-solvent ifneeded) to determine the origin of the selectivity observed for theanalytes of interest by embodiments of the disclosed NIR dyes.Conjugates, adducts or reaction products may be isolated andcharacterized by HPLC and LC-MS ESI. Crystal structures of isolatedcomplexes, adducts or reaction products may be determined by X-Raycrystallography. Binding constants for complex formation between theanalytes of interest and NIR fluorescent probes may be obtained usingspectrometric methods including: 1H NMR, 2D 1H NMR (ROESY, NOESY),UV-vis or fluorescence.

Selectivity and crossreactivity may be evaluated in the presence ofpossible interferences (e.g., Cys, Cys-Gly, and/or Cys-Glu for GSH).Influence of factors involved in selectivity/specificity including pH,ionic strength, type of buffering system, and molar ratios ofanalyte:NIR fluorophore-viologen conjugate may be evaluated usingspectrometric methodologies.

Example 8 Selective Detection of SAICAr and S-Ado in Urine

Embodiments of the disclosed NIR dyes functionalized with at least oneboronic acid functional group (i.e., —NHR^(c) where R^(c) is as definedabove) may serve as indicators for ADLS deficiency. Mechanisticinvestigations and characterization studies similar to those performedin Example 5 may be used to determine selectivity for SAICAr and/orS-Ado detection. Promising candidates may be evaluated in urine and/orsynthetic urine samples.

Additional patent documents describing subject matter or backgroundinformation which may be pertinent to the present disclosure includeU.S. Publication No. 2008/0261315, U.S. Publication No. 2010/0051826,and WO 2008/011508, each of which is incorporated in its entirety hereinby reference.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A compound having a chemical structure selected from


2. A kit for detecting an analyte, comprising at least one compoundaccording to claim 1, wherein the compound when combined with a samplecomprising the analyte will undergo a change in its absorbance spectrum,emission spectrum, or both compared to the compound in a sample thatdoes not comprise the analyte.
 3. The kit of claim 2, further comprisingat least one buffer solution in which the compound when combined with asample comprising the analyte will undergo a change in its absorbancespectrum, emission spectrum, or both compared to the compound combinedwith the buffer solution and a sample that does not comprise theanalyte.
 4. The kit of claim 2, where the analyte comprises glutathione,cysteine, homocysteine, succinyl-5-amino-4-imidazolecarboxamideriboside, succinyladenosine, or a combination ofsuccinyl-5-amino-4-imidazolecarboxamide riboside and succinyladenosine.5. The kit of claim 2, further comprising a plurality of disposablecontainers in which a reaction between the compound and the analyte canbe performed.
 6. The kit of claim 5, where an amount of the compoundeffective to undergo a detectable change in the absorbance spectrum, theemission spectrum, or both when reacted with the analyte is premeasuredinto the plurality of disposable containers.
 7. A method for selectivelydetecting an analyte in a biological fluid, comprising: (a) combining acompound according to general formula (III) or (IV) with a biologicalfluid to form a solution

where each bond depicted as “

” is a single or double bond as needed to satisfy valence requirements;X¹ is O, S, or N(H); R⁶ is hydroxyl, amino, alkyl amino, or —NHR^(c)when the bond between R⁶ and ring A is a single bond, or R⁶ is oxygen,imino, iminium, alkyl imino, or alkyl iminium when the bond between R⁶and ring A is a double bond, where R^(C) is

R⁷ is hydrogen or halogen; R⁹-R¹² independently are hydrogen, loweralkyl, carboxyl, amino, or —SO₃H; R¹³ is hydrogen, lower alkyl, loweralkoxy, —SO₃H or —COOR¹⁴ where R¹⁴ is hydrogen or lower alkyl and thebond depicted as “

” in ring B is a double bond, or R¹³ is one or more atoms forming a ringsystem with rings B and D and the bond depicted as “

” in ring B is a single bond; R¹⁶ and R¹⁸ independently are hydrogen,hydroxyl, thiol, oxygen, lower alkoxy, amino, alkyl amino, or —NHR^(c),and at least one of R¹⁶ and R¹⁸ is other than hydrogen; R¹⁹ and R²¹independently are hydrogen, hydroxyl, thiol, oxygen, amino, alkyl amino,imino, iminium, alkyl imino, alkyl iminium, or —NHR^(c) where R^(c) isas defined above, and at least one of R¹⁹ and R²¹ is other thanhydrogen; and wherein if X¹ is oxygen in general formula (III), then R⁶is other than oxygen or alkyl amino, or R¹³ is other than one or moreatoms forming a ring system with rings B and D, or R¹⁶ is other thanhydroxyl or hydrogen, or R¹⁸ is other than hydroxyl or hydrogen, or atleast one of R⁷, R⁹, R¹⁰, R¹¹, R¹², R¹³ is other than hydrogen, or ifthe compound has a chemical structure according to general formula (IV),then at least one of R⁹-R¹³, R¹⁶, R¹⁸, R¹⁹, or R²¹is other thanhydrogen, hydroxyl, halogen, oxygen, lower alkyl, amino, or thiol, orR¹³ is one or more atoms forming a ring system with rings B and D andthe bond depicted as “

” in ring B is a single bond; (b) exposing the solution to a lightsource; and (c) detecting the analyte by detecting fluorescence from thecompound, wherein the analyte comprises cysteine, homocysteine,glutathione, succinyl-5-amino-4-imidazolecarboxamide riboside,succinyladenosine, or a combination thereof.
 8. The method of claim 7where detecting fluorescence from the compound comprises detectingfluorescence at a wavelength corresponding to an emission spectrummaximum of the compound.
 9. The method of claim 8, further comprisingquantitating the analyte by measuring an amount of fluorescence from thecompound at a wavelength corresponding to an emission spectrum maximumof the compound.
 10. The method of claim 7 where the biological fluidcomprises blood or urine.